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Sample records for nucleotides regulate pollen

  1. Intersection of two signalling pathways: extracellular nucleotides regulate pollen germination and pollen tube growth via nitric oxide.

    PubMed

    Reichler, Stuart A; Torres, Jonathan; Rivera, Amy L; Cintolesi, Viviana A; Clark, Greg; Roux, Stanley J

    2009-01-01

    Plant and animal cells release or secrete ATP by various mechanisms, and this activity allows extracellular ATP to serve as a signalling molecule. Recent reports suggest that extracellular ATP induces plant responses ranging from increased cytosolic calcium to changes in auxin transport, xenobiotic resistance, pollen germination, and growth. Although calcium has been identified as a secondary messenger for the extracellular ATP signal, other parts of this signal transduction chain remain unknown. Increasing the extracellular concentration of ATPgammaS, a poorly-hydrolysable ATP analogue, inhibited both pollen germination and pollen tube elongation, while the addition of AMPS had no effect. Because pollen tube elongation is also sensitive to nitric oxide, this raised the possibility that a connection exists between the two pathways. Four approaches were used to test whether the germination and growth effects of extracellular ATPgammaS were transduced via nitric oxide. The results showed that increases in extracellular ATPgammaS induced increases in cellular nitric oxide, chemical agonists of the nitric oxide signalling pathway lowered the threshold of extracellular ATPgammaS that inhibits pollen germination, an antagonist of guanylate cyclase, which can inhibit some nitric oxide signalling pathways, blocked the ATPgammaS-induced inhibition of both pollen germination and pollen tube elongation, and the effects of applied ATPgammaS were blocked in nia1nia2 mutants, which have diminished NO production. The concurrence of these four data sets support the conclusion that the suppression of pollen germination and pollen tube elongation by extracellular nucleotides is mediated in part via the nitric oxide signalling pathway.

  2. A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important Role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant-sp...

  3. A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana

    USDA-ARS?s Scientific Manuscript database

    Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant sp...

  4. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis.

    PubMed

    Gao, Qi-Fei; Gu, Li-Li; Wang, Hui-Qin; Fei, Cui-Fang; Fang, Xiang; Hussain, Jamshaid; Sun, Shu-Jing; Dong, Jing-Yun; Liu, Hongtao; Wang, Yong-Fei

    2016-03-15

    In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca(2+) gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca(2+) channels in pollen tube tips are core components that regulate Ca(2+) gradients by mediating and regulating external Ca(2+) influx. Therefore, Ca(2+) channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca(2+) channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca(2+) gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/-) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18's transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca(2+) channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca(2+) channel for pollen tube guidance in Arabidopsis.

  5. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis

    PubMed Central

    Gao, Qi-Fei; Gu, Li-Li; Wang, Hui-Qin; Fei, Cui-Fang; Fang, Xiang; Hussain, Jamshaid; Sun, Shu-Jing; Dong, Jing-Yun; Liu, Hongtao; Wang, Yong-Fei

    2016-01-01

    In flowering plants, pollen tubes are guided into ovules by multiple attractants from female gametophytes to release paired sperm cells for double fertilization. It has been well-established that Ca2+ gradients in the pollen tube tips are essential for pollen tube guidance and that plasma membrane Ca2+ channels in pollen tube tips are core components that regulate Ca2+ gradients by mediating and regulating external Ca2+ influx. Therefore, Ca2+ channels are the core components for pollen tube guidance. However, there is still no genetic evidence for the identification of the putative Ca2+ channels essential for pollen tube guidance. Here, we report that the point mutations R491Q or R578K in cyclic nucleotide-gated channel 18 (CNGC18) resulted in abnormal Ca2+ gradients and strong pollen tube guidance defects by impairing the activation of CNGC18 in Arabidopsis. The pollen tube guidance defects of cngc18-17 (R491Q) and of the transfer DNA (T-DNA) insertion mutant cngc18-1 (+/−) were completely rescued by CNGC18. Furthermore, domain-swapping experiments showed that CNGC18’s transmembrane domains are indispensable for pollen tube guidance. Additionally, we found that, among eight Ca2+ channels (including six CNGCs and two glutamate receptor-like channels), CNGC18 was the only one essential for pollen tube guidance. Thus, CNGC18 is the long-sought essential Ca2+ channel for pollen tube guidance in Arabidopsis. PMID:26929345

  6. Regulation of Pollen Tube Growth by Transglutaminase

    PubMed Central

    Cai, Giampiero; Serafini-Fracassini, Donatella; Del Duca, Stefano

    2013-01-01

    In pollen tubes, cytoskeleton proteins are involved in many aspects of pollen germination and growth, from the transport of sperm cells to the asymmetrical distribution of organelles to the deposition of cell wall material. These activities are based on the dynamics of the cytoskeleton. Changes to both actin filaments and microtubules are triggered by specific proteins, resulting in different organization levels suitable for the different functions of the cytoskeleton. Transglutaminases are enzymes ubiquitous in all plant organs and cell compartments. They catalyze the post-translational conjugation of polyamines to different protein targets, such as the cytoskeleton. Transglutaminases are suggested to have a general role in the interaction between pollen tubes and the extracellular matrix during fertilization and a specific role during the self-incompatibility response. In such processes, the activity of transglutaminases is enhanced, leading to the formation of cross-linked products (including aggregates of tubulin and actin). Consequently, transglutaminases are suggested to act as regulators of cytoskeleton dynamics. The distribution of transglutaminases in pollen tubes is affected by both membrane dynamics and the cytoskeleton. Transglutaminases are also secreted in the extracellular matrix, where they may take part in the assembly and/or strengthening of the pollen tube cell wall. PMID:27137368

  7. A pollen-specific RALF from tomato that regulates pollen tube elongation.

    PubMed

    Covey, Paul A; Subbaiah, Chalivendra C; Parsons, Ronald L; Pearce, Gregory; Lay, Fung T; Anderson, Marilyn A; Ryan, Clarence A; Bedinger, Patricia A

    2010-06-01

    Rapid Alkalinization Factors (RALFs) are plant peptides that rapidly increase the pH of plant suspension cell culture medium and inhibit root growth. A pollen-specific tomato (Solanum lycopersicum) RALF (SlPRALF) has been identified. The SlPRALF gene encodes a preproprotein that appears to be processed and released from the pollen tube as an active peptide. A synthetic SlPRALF peptide based on the putative active peptide did not affect pollen hydration or viability but inhibited the elongation of normal pollen tubes in an in vitro growth system. Inhibitory effects of SlPRALF were detectable at concentrations as low as 10 nm, and complete inhibition was observed at 1 mum peptide. At least 10-fold higher levels of alkSlPRALF, which lacks disulfide bonds, were required to see similar effects. A greater effect of peptide was observed in low-pH-buffered medium. Inhibition of pollen tube elongation was reversible if peptide was removed within 15 min of exposure. Addition of 100 nm SlPRALF to actively growing pollen tubes inhibited further elongation until tubes were 40 to 60 mum in length, after which pollen tubes became resistant to the peptide. The onset of resistance correlated with the timing of the exit of the male germ unit from the pollen grain into the tube. Thus, exogenous SlPRALF acts as a negative regulator of pollen tube elongation within a specific developmental window.

  8. Regulation of pollen tube polarity: Feedback loops rule

    USDA-ARS?s Scientific Manuscript database

    Targeted delivery of immotile sperm through growing pollen tubes is a crucial step in achieving sexual reproduction in angiosperms. Unlike diffuse-growing cells, the growth of a pollen tube is restricted to the very apical region where targeted exocytosis and regulated endocytosis occur. The plant-s...

  9. Honey loading for pollen collection: regulation of crop content in honeybee pollen foragers on leaving hive

    NASA Astrophysics Data System (ADS)

    Harano, Ken-ichi; Mitsuhata-Asai, Akiko; Sasaki, Masami

    2014-07-01

    Before foraging honeybees leave the hive, each bee loads its crop with some amount of honey "fuel" depending on the distance to the food source and foraging experience. For pollen collection, there is evidence that foragers carry additional honey as "glue" to build pollen loads. This study examines whether pollen foragers of the European honeybee Apis mellifera regulate the size of the crop load according to food-source distances upon leaving the hive and how foraging experience affects load regulation. The crop contents of bees foraging on crape myrtle Lagerstroemia indica, which has no nectary, were larger than those foraging on nectar from other sources, confirming a previous finding that pollen foragers carry glue in addition to fuel honey from the hive. Crop contents of both waggle dancers and dance followers showed a significant positive correlation with waggle-run durations. These results suggest that bees carry a distance-dependent amount of fuel honey in addition to a fixed amount of glue honey. Crop contents on leaving the hive were statistically larger in dancers than followers. Based on these results, we suggest that pollen foragers use information obtained through foraging experience to adjust crop contents on leaving the hive.

  10. Honey loading for pollen collection: regulation of crop content in honeybee pollen foragers on leaving hive.

    PubMed

    Harano, Ken-ichi; Mitsuhata-Asai, Akiko; Sasaki, Masami

    2014-07-01

    Before foraging honeybees leave the hive, each bee loads its crop with some amount of honey "fuel" depending on the distance to the food source and foraging experience. For pollen collection, there is evidence that foragers carry additional honey as "glue" to build pollen loads. This study examines whether pollen foragers of the European honeybee Apis mellifera regulate the size of the crop load according to food-source distances upon leaving the hive and how foraging experience affects load regulation. The crop contents of bees foraging on crape myrtle Lagerstroemia indica, which has no nectary, were larger than those foraging on nectar from other sources, confirming a previous finding that pollen foragers carry glue in addition to fuel honey from the hive. Crop contents of both waggle dancers and dance followers showed a significant positive correlation with waggle-run durations. These results suggest that bees carry a distance-dependent amount of fuel honey in addition to a fixed amount of glue honey. Crop contents on leaving the hive were statistically larger in dancers than followers. Based on these results, we suggest that pollen foragers use information obtained through foraging experience to adjust crop contents on leaving the hive.

  11. Regulation of innate immunity by extracellular nucleotides

    PubMed Central

    Gorini, Stefania; Gatta, Lucia; Pontecorvo, Laura; Vitiello, Laura; la Sala, Andrea

    2013-01-01

    Extracellular ATP (eATP) is the most abundant among extracellular nucleotides and is commonly considered as a classical danger signal, which stimulates immune responses in the presence of tissue injury. In fact, increased nucleotide concentration in the extracellular space is generally closely associated with tissue stress or damage. However non-lytic nucleotide release may also occur in many cell types under a variety of conditions. Extracellular nucleotides are sensed by a class of plasma membrane receptors called P2 purinergic receptors (P2Rs). P2 receptors are expressed by all immunological cells and their activation elicits different responses. Extracellular ATP can act as an initiator or terminator of immune responses being able to induce different effects on immune cells depending on the pattern of P2 receptors engaged, the duration of the stimulus and its concentration in the extracellular milieu. Millimolar (high) concentrations of extracellular ATP, induce predominantly proinflammatory effects, while micromolar (low) doses exert mainly tolerogenic/immunosuppressive action. Moreover small, but significant differences in the pattern of P2 receptor expression in mice and humans confer diverse capacities of ATP in regulating the immune response. PMID:23358447

  12. PCP-B class pollen coat proteins are key regulators of the hydration checkpoint in Arabidopsis thaliana pollen-stigma interactions.

    PubMed

    Wang, Ludi; Clarke, Lisa A; Eason, Russell J; Parker, Christopher C; Qi, Baoxiu; Scott, Rod J; Doughty, James

    2017-01-01

    The establishment of pollen-pistil compatibility is strictly regulated by factors derived from both male and female reproductive structures. Highly diverse small cysteine-rich proteins (CRPs) have been found to play multiple roles in plant reproduction, including the earliest stages of the pollen-stigma interaction. Secreted CRPs found in the pollen coat of members of the Brassicaceae, the pollen coat proteins (PCPs), are emerging as important signalling molecules that regulate the pollen-stigma interaction. Using a combination of protein characterization, expression and phylogenetic analyses we identified a novel class of Arabidopsis thaliana pollen-borne CRPs, the PCP-Bs (for pollen coat protein B-class) that are related to embryo surrounding factor (ESF1) developmental regulators. Single and multiple PCP-B mutant lines were utilized in bioassays to assess effects on pollen hydration, adhesion and pollen tube growth. Our results revealed that pollen hydration is severely impaired when multiple PCP-Bs are lost from the pollen coat. The hydration defect also resulted in reduced pollen adhesion and delayed pollen tube growth in all mutants studied. These results demonstrate that AtPCP-Bs are key regulators of the hydration 'checkpoint' in establishment of pollen-stigma compatibility. In addition, we propose that interspecies diversity of PCP-Bs may contribute to reproductive barriers in the Brassicaceae. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  13. A developmentally regulated MAP kinase activated by hydration in tobacco pollen.

    PubMed Central

    Wilson, C; Voronin, V; Touraev, A; Vicente, O; Heberle-Bors, E

    1997-01-01

    A novel mitogen-activated protein (MAP) kinase signaling pathway has been identified in tobacco. This pathway is developmentally regulated during pollen maturation and is activated by hydration during pollen germination. Analysis of different stages of pollen development showed that transcriptional and translational induction of MAP kinase synthesis occurs at the mid-bicellular stage of pollen maturation. However, the MAP kinase is stored in an inactive form in the mature, dry pollen grain. Kinase activation is very rapid after hydration of the dry pollen, peaking at approximately 5 min and decreasing thereafter. Immunoprecipitation of the kinase activity by an anti-phosphotyrosine antibody is consistent with the activation of a MAP kinase. The kinetics of activation suggest that the MAP kinase plays a role in the activation of the pollen grain after hydration rather than in pollen tube growth. PMID:9401129

  14. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules

    PubMed Central

    Sesma, Juliana I.; Kreda, Silvia M.; Okada, Seiko F.; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C.; O'Neal, Wanda K.; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori

    2013-01-01

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca2+-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins. PMID:23467297

  15. Vesicular nucleotide transporter regulates the nucleotide content in airway epithelial mucin granules.

    PubMed

    Sesma, Juliana I; Kreda, Silvia M; Okada, Seiko F; van Heusden, Catharina; Moussa, Lama; Jones, Lisa C; O'Neal, Wanda K; Togawa, Natsuko; Hiasa, Miki; Moriyama, Yoshinori; Lazarowski, Eduardo R

    2013-05-15

    Nucleotides within the airway surface liquid promote fluid secretion via activation of airway epithelial purinergic receptors. ATP is stored within and released from mucin granules as co-cargo with mucins, but the mechanism by which ATP, and potentially other nucleotides, enter the lumen of mucin granules is not known. We assessed the contribution of the recently identified SLC17A9 vesicle nucleotide transporter (VNUT) to the nucleotide availability within isolated mucin granules and further examined the involvement of VNUT in mucin granule secretion-associated nucleotide release. RT-PCR and Western blot analyses indicated that VNUT is abundantly expressed in airway epithelial goblet-like Calu-3 cells, migrating as a duplex with apparent mobility of 55 and 60 kDa. Subcellular fractionation studies indicated that VNUT55 was associated with high-density mucin granules, whereas VNUT60 was associated with low-density organelles. Immunofluorescence studies showed that recombinant VNUT localized to mucin granules and other organelles. Mucin granules isolated from VNUT short hairpin RNA-expressing cells exhibited a marked reduction of ATP, ADP, AMP, and UTP levels within granules. Ca(2+)-regulated vesicular ATP release was markedly reduced in these cells, but mucin secretion was not affected. These results suggest that VNUT is the relevant nucleotide transporter responsible for the uptake of cytosolic nucleotides into mucin granules. By controlling the entry of nucleotides into mucin granules, VNUT contributes to the release of purinergic signaling molecules necessary for the proper hydration of co-released mucins.

  16. Arabidopsis RIC1 Severs Actin Filaments at the Apex to Regulate Pollen Tube Growth

    PubMed Central

    Zhou, Zhenzhen; Shi, Haifan; Chen, Binqing; Zhang, Ruihui; Huang, Shanjin; Fu, Ying

    2015-01-01

    Pollen tubes deliver sperms to the ovule for fertilization via tip growth. The rapid turnover of F-actin in pollen tube tips plays an important role in this process. In this study, we demonstrate that Arabidopsis thaliana RIC1, a member of the ROP-interactive CRIB motif-containing protein family, regulates pollen tube growth via its F-actin severing activity. Knockout of RIC1 enhanced pollen tube elongation, while overexpression of RIC1 dramatically reduced tube growth. Pharmacological analysis indicated that RIC1 affected F-actin dynamics in pollen tubes. In vitro biochemical assays revealed that RIC1 directly bound and severed F-actin in the presence of Ca2+ in addition to interfering with F-actin turnover by capping F-actin at the barbed ends. In vivo, RIC1 localized primarily to the apical plasma membrane (PM) of pollen tubes. The level of RIC1 at the apical PM oscillated during pollen tube growth. The frequency of F-actin severing at the apex was notably decreased in ric1-1 pollen tubes but was increased in pollen tubes overexpressing RIC1. We propose that RIC1 regulates F-actin dynamics at the apical PM as well as the cytosol by severing F-actin and capping the barbed ends in the cytoplasm, establishing a novel mechanism that underlies the regulation of pollen tube growth. PMID:25804540

  17. Organization and regulation of the actin cytoskeleton in the pollen tube

    PubMed Central

    Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Liu, Xiaonan; Zhang, Ruihui; Huang, Shanjin

    2015-01-01

    Proper organization of the actin cytoskeleton is crucial for pollen tube growth. However, the precise mechanisms by which the actin cytoskeleton regulates pollen tube growth remain to be further elucidated. The functions of the actin cytoskeleton are dictated by its spatial organization and dynamics. However, early observations of the distribution of actin filaments at the pollen tube apex were quite perplexing, resulting in decades of controversial debate. Fortunately, due to improvements in fixation regimens for staining actin filaments in fixed pollen tubes, as well as the adoption of appropriate markers for visualizing actin filaments in living pollen tubes, this issue has been resolved and has given rise to the consensus view of the spatial distribution of actin filaments throughout the entire pollen tube. Importantly, recent descriptions of the dynamics of individual actin filaments in the apical region have expanded our understanding of the function of actin in regulation of pollen tube growth. Furthermore, careful documentation of the function and mode of action of several actin-binding proteins expressed in pollen have provided novel insights into the regulation of actin spatial distribution and dynamics. In the current review, we summarize our understanding of the organization, dynamics, and regulation of the actin cytoskeleton in the pollen tube. PMID:25620974

  18. Arabidopsis FIM5 decorates apical actin filaments and regulates their organization in the pollen tube

    PubMed Central

    Zhang, Meng; Zhang, Ruihui; Qu, Xiaolu; Huang, Shanjin

    2016-01-01

    The actin cytoskeleton is increasingly recognized as a major regulator of pollen tube growth. Actin filaments have distinct distribution patterns and dynamic properties within different regions of the pollen tube. Apical actin filaments are highly dynamic and crucial for pollen tube growth. However, how apical actin filaments are generated and properly constructed remains an open question. Here we showed that Arabidopsis fimbrin5 (FIM5) decorates filamentous structures throughout the entire tube but is apically concentrated. Apical actin structures are disorganized to different degrees in the pollen tubes of fim5 loss-of-function mutants. Further observations suggest that apical actin structures are not constructed properly because apical actin filaments cannot be maintained at the cortex of fim5 pollen tubes. Actin filaments appeared to be more curved in fim5 pollen tubes and this was confirmed by measurements showing that the convolutedness and the rate of change of convolutedness of actin filaments was significantly increased in fim5 pollen tubes. This suggests that the rigidity of the actin filaments may be compromised in fim5 pollen tubes. Further, the apical cell wall composition is altered, implying that tip-directed vesicle trafficking events are impaired in fim5 pollen tubes. Thus, we found that FIM5 decorates apical actin filaments and regulates their organization in order to drive polarized pollen tube growth. PMID:27117336

  19. Regulation of the Pollen-Specific Actin-Depolymerizing Factor LlADF1

    PubMed Central

    Allwood, Ellen G.; Anthony, Richard G.; Smertenko, Andrei P.; Reichelt, Stefanie; Drobak, Bjorn K.; Doonan, John H.; Weeds, Alan G.; Hussey, Patrick J.

    2002-01-01

    Pollen tube growth is dependent on a dynamic actin cytoskeleton, suggesting that actin-regulating proteins are involved. We have examined the regulation of the lily pollen-specific actin-depolymerizing factor (ADF) LlADF1. Its actin binding and depolymerizing activity is pH sensitive, inhibited by certain phosphoinositides, but not controlled by phosphorylation. Compared with its F-actin binding properties, its low activity in depolymerization assays has been used to explain why pollen ADF decorates F-actin in pollen grains. This low activity is incompatible with a role in increasing actin dynamics necessary to promote pollen tube growth. We have identified a plant homolog of actin-interacting protein, AIP1, which enhances the depolymerization of F-actin in the presence of LlADF1 by ∼60%. Both pollen ADF and pollen AIP1 bind F-actin in pollen grains but are mainly cytoplasmic in pollen tubes. Our results suggest that together these proteins remodel actin filaments as pollen grains enter and exit dormancy. PMID:12417710

  20. Profilin Regulates Apical Actin Polymerization to Control Polarized Pollen Tube Growth.

    PubMed

    Liu, Xiaonan; Qu, Xiaolu; Jiang, Yuxiang; Chang, Ming; Zhang, Ruihui; Wu, Youjun; Fu, Ying; Huang, Shanjin

    2015-12-07

    Pollen tube growth is an essential step during flowering plant reproduction, whose growth depends on a population of dynamic apical actin filaments. Apical actin filaments were thought to be involved in the regulation of vesicle fusion and targeting in the pollen tube. However, the molecular mechanisms that regulate the construction of apical actin structures in the pollen tube remain largely unclear. Here, we identify profilin as an important player in the regulation of actin polymerization at the apical membrane in the pollen tube. Downregulation of profilin decreased the amount of filamentous actin and induced disorganization of apical actin filaments, and reduced tip-directed vesicle transport and accumulation in the pollen tube. Direct visualization of actin dynamics revealed that the elongation of actin filaments originating at the apical membrane decreased in profilin mutant pollen tubes. Mutant profilin that is defective in binding poly-L-proline only partially rescues the actin polymerization defect in profilin mutant pollen tubes, although it fully rescues the actin turnover phenotype. We propose that profilin controls the construction of actin structures at the pollen tube tip, presumably by favoring formin-mediated actin polymerization at the apical membrane.

  1. Cyclic nucleotide-regulated ion channels: spotlight on symmetry.

    PubMed

    Taraska, Justin W; Zagotta, William N

    2007-09-01

    In this issue of Structure, Chiu et al. (2007) report the 16 A EM structure of the prokaryotic cyclic nucleotide-regulated K(+) channel MloK1. This structure reveals that the channel is arranged as a four-fold symmetric tetramer.

  2. Arabidopsis thaliana CML25 mediates the Ca(2+) regulation of K(+) transmembrane trafficking during pollen germination and tube elongation.

    PubMed

    Wang, Shuang-Shuang; Diao, Wen-Zhu; Yang, Xue; Qiao, Zhu; Wang, Mei; Acharya, Biswa R; Zhang, Wei

    2015-11-01

    The concentration alteration of cytosolic-free calcium ([Ca(2+) ]cyt ) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca(2+) -binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K(+) and Ca(2+) concentration than wild-type pollen. The disruption of CML25 increased the [Ca(2+) ]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca(2+) -dependent inhibition of whole-cell inward K(+) currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca(2+) -mediated regulation of K(+) influx during pollen germination and tube elongation. © 2015 John Wiley & Sons Ltd.

  3. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen.

    PubMed

    Gao, Xin-Qi; Liu, Chang Zhen; Li, Dan Dan; Zhao, Ting Ting; Li, Fei; Jia, Xiao Na; Zhao, Xin-Ying; Zhang, Xian Sheng

    2016-07-01

    Pollen-stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen-stigma interactions is poorly understood. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinβγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinβγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINβγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen-stigma interactions during pollination.

  4. Cardiac Cyclic Nucleotide Phosphodiesterases: Function, Regulation, and Therapeutic Prospects

    PubMed Central

    Knight, W. E.; Yan, C.

    2014-01-01

    The second messengers cAMP and cGMP exist in multiple discrete compartments and regulate a variety of biological processes in the heart. The cyclic nucleotide phosphodiesterases, by catalyzing the hydrolysis of cAMP and cGMP, play crucial roles in controlling the amplitude, duration, and compartmentalization of cyclic nucleotide signaling. Over 60 phosphodiesterase isoforms, grouped into 11 families, have been discovered to date. In the heart, both cAMP- and cGMP-hydrolyzing phosphodiesterases play important roles in physiology and pathology. At least 7 of the 11 phosphodiesterase family members appear to be expressed in the myocardium, and evidence supports phosphodiesterase involvement in regulation of many processes important for normal cardiac function including pacemaking and contractility, as well as many pathological processes including remodeling and myocyte apoptosis. Pharmacological inhibitors for a number of phosphodiesterase families have also been used clinically or preclinically to treat several types of cardiovascular disease. In addition, phosphodiesterase inhibitors are also being considered for treatment of many forms of disease outside the cardiovascular system, raising the possibility of cardiovascular side effects of such agents. This review will discuss the roles of phosphodiesterases in the heart, in terms of expression patterns, regulation, and involvement in physiological and pathological functions. Additionally, the cardiac effects of various phosphodiesterase inhibitors, both potentially beneficial and detrimental, will be discussed. PMID:22951903

  5. Pho5p and Newly Identified Nucleotide Pyrophosphatases/ Phosphodiesterases Regulate Extracellular Nucleotide Phosphate Metabolism in Saccharomyces cerevisiae

    PubMed Central

    Kennedy, Eileen J.; Pillus, Lorraine; Ghosh, Gourisankar

    2005-01-01

    Extracellular nucleotides play many biological roles, including intercellular communication and modulation of nucleotide receptor signaling, and are dependent on the phosphorylation state of the nucleotide. Regulation of nucleotide phosphorylation is necessary, and a specialized class of enzymes, nucleotide pyrophosphatases/phosphodiesterases (E-NPPs), has been identified in mammals to perform this function. Although the E-NPP class is conserved among complex eukaryotes, this system has not yet been identified in Saccharomyces cerevisiae. Using genetic and biochemical experiments, we show that two orthologs of the E-NPP family, referred to as Npp1p and Npp2p, exist in budding yeast and can perform nucleotide phosphate hydrolysis. This activity is enhanced during phosphate starvation, where hydrolyzed phosphates can be imported from extracellular sources and utilized to overcome phosphate starvation through the activity of the Pho5p acid phosphatase. The added compensatory effect by Pho5p is also a newly established role for Pho5p. This study demonstrates that extracellular nucleotide phosphate metabolism appears to be controlled by at least two independent regulatory mechanisms, uniting phosphate starvation with extracellular nucleotide regulation. PMID:16278456

  6. Arabidopsis JINGUBANG Is a Negative Regulator of Pollen Germination That Prevents Pollination in Moist Environments[OPEN

    PubMed Central

    Ma, Fei; Zhu, Qiao-Yun; Zhang, Quan; Sodmergen

    2016-01-01

    The molecular mechanism of pollen germination and pollen tube growth has been revealed in detail during the last decade, while the mechanism that suspends pollen grains in a dormant state is largely unclear. Here, we identified the JINGUBANG (JGB) gene by screening pollen-specific genes for those that are unnecessary for pollen germination. We showed that the pollen of the jgb loss-of-function mutant exhibited hyperactive germination in sucrose-only medium and inside the anther, while this phenotype was rescued by the transgenic expression of JGB in jgb plants. JGB contains seven WD40 repeats and is highly conserved in flowering plants. Overexpression of JGB inhibits pollen germination. These results indicate that JGB is a novel negative regulator of pollen germination. In addition, we found that jasmonic acid (JA) abundance was significantly elevated in jgb pollen, while exogenous application of methyl jasmonate rescued the inhibition of pollen germination in plants overexpressing JGB. Based on the molecular features of JGB and on the finding that it interacts with a known JA biosynthesis-related transcription factor, TCP4, we propose that JGB, together with TCP4, forms a regulatory complex that controls pollen JA synthesis, ensuring pollination in moist environments. PMID:27468890

  7. Biphasic regulation of the transcription factor ABORTED MICROSPORES (AMS) is essential for tapetum and pollen development in Arabidopsis.

    PubMed

    Ferguson, Alison C; Pearce, Simon; Band, Leah R; Yang, Caiyun; Ferjentsikova, Ivana; King, John; Yuan, Zheng; Zhang, Dabing; Wilson, Zoe A

    2017-01-01

    Viable pollen is essential for plant reproduction and crop yield. Its production requires coordinated expression at specific stages during anther development, involving early meiosis-associated events and late pollen wall formation. The ABORTED MICROSPORES (AMS) transcription factor is a master regulator of sporopollenin biosynthesis, secretion and pollen wall formation in Arabidopsis. Here we show that it has complex regulation and additional essential roles earlier in pollen formation. An inducible-AMS reporter was created for functional rescue, protein expression pattern analysis, and to distinguish between direct and indirect targets. Mathematical modelling was used to create regulatory networks based on wild-type RNA and protein expression. Dual activity of AMS was defined by biphasic protein expression in anther tapetal cells, with an initial peak around pollen meiosis and then later during pollen wall development. Direct AMS-regulated targets exhibit temporal regulation, indicating that additional factors are associated with their regulation. We demonstrate that AMS biphasic expression is essential for pollen development, and defines distinct functional activities during early and late pollen development. Mathematical modelling suggests that AMS may competitively form a protein complex with other tapetum-expressed transcription factors, and that biphasic regulation is due to repression of upstream regulators and promotion of AMS protein degradation.

  8. Arabidopsis ACTIN-DEPOLYMERIZING FACTOR7 Severs Actin Filaments and Regulates Actin Cable Turnover to Promote Normal Pollen Tube Growth[W

    PubMed Central

    Zheng, Yiyan; Xie, Yurong; Jiang, Yuxiang; Qu, Xiaolu; Huang, Shanjin

    2013-01-01

    Actin filaments are often arranged into higher-order structures, such as the longitudinal actin cables that generate the reverse fountain cytoplasmic streaming pattern present in pollen tubes. While several actin binding proteins have been implicated in the generation of these cables, the mechanisms that regulate their dynamic turnover remain largely unknown. Here, we show that Arabidopsis thaliana ACTIN-DEPOLYMERIZING FACTOR7 (ADF7) is required for turnover of longitudinal actin cables. In vitro biochemical analyses revealed that ADF7 is a typical ADF that prefers ADP-G-actin over ATP-G-actin. ADF7 inhibits nucleotide exchange on actin and severs filaments, but its filament severing and depolymerizing activities are less potent than those of the vegetative ADF1. ADF7 primarily decorates longitudinal actin cables in the shanks of pollen tubes. Consistent with this localization pattern, the severing frequency and depolymerization rate of filaments significantly decreased, while their maximum lifetime significantly increased, in adf7 pollen tube shanks. Furthermore, an ADF7–enhanced green fluorescent protein fusion with defective severing activity but normal G-actin binding activity could not complement adf7, providing compelling evidence that the severing activity of ADF7 is vital for its in vivo functions. These observations suggest that ADF7 evolved to promote turnover of longitudinal actin cables by severing actin filaments in pollen tubes. PMID:24058157

  9. MAP18 regulates the direction of pollen tube growth in Arabidopsis by modulating F-actin organization.

    PubMed

    Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

    2013-03-01

    For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca(2+)-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments.

  10. Regulation of endonuclease activity in human nucleotide excision repair

    PubMed Central

    Fagbemi, Adebanke F.; Orelli, Barbara; Schärer, Orlando D.

    2011-01-01

    Nucleotide excision repair (NER) is a DNA repair pathway that is responsible for removing a variety of lesions caused by harmful UV light, chemical carcinogens, and environmental mutagens from DNA. NER involves the concerted action of over 30 proteins that sequentially recognize a lesion, excise it in the form of an oligonucleotide, and fill in the resulting gap by repair synthesis. ERCC1-XPF and XPG are structure-specific endonucleases responsible for carrying out the incisions 5′ and 3′ to the damage respectively, culminating in the release of the damaged oligonucleotide. This review focuses on the recent work that led to a greater understanding of how the activities of ERCC1-XPF and XPG are regulated in NER to prevent unwanted cuts in DNA or the persistence of gaps after incision that could result in harmful, cytotoxic DNA structures. PMID:21592868

  11. Salicylic Acid Regulates Pollen Tip Growth through an NPR3/NPR4-Independent Pathway.

    PubMed

    Rong, Duoyan; Luo, Nan; Mollet, Jean Claude; Liu, Xuanming; Yang, Zhenbiao

    2016-11-07

    Tip growth is a common strategy for the rapid elongation of cells to forage the environment and/or to target to long-distance destinations. In the model tip growth system of Arabidopsis pollen tubes, several small-molecule hormones regulate their elongation, but how these rapidly diffusing molecules control extremely localized growth remains mysterious. Here we show that the interconvertible salicylic acid (SA) and methylated SA (MeSA), well characterized for their roles in plant defense, oppositely regulate Arabidopsis pollen tip growth with SA being inhibitory and MeSA stimulatory. The effect of SA and MeSA was independent of known NPR3/NPR4 SA receptor-mediated signaling pathways. SA inhibited clathrin-mediated endocytosis in pollen tubes associated with an increased accumulation of less stretchable demethylated pectin in the apical wall, whereas MeSA did the opposite. Furthermore, SA and MeSA alter the apical activation of ROP1 GTPase, a key regulator of tip growth in pollen tubes, in an opposite manner. Interestingly, both MeSA methylesterase and SA methyltransferase, which catalyze the interconversion between SA and MeSA, are localized at the apical region of pollen tubes, indicating of the tip-localized production of SA and MeSA and consistent with their effects on the apical cellular activities. These findings suggest that local generation of a highly diffusible signal can regulate polarized cell growth, providing a novel mechanism of cell polarity control apart from the one involving protein and mRNA polarization.

  12. The Arabidopsis KINβγ Subunit of the SnRK1 Complex Regulates Pollen Hydration on the Stigma by Mediating the Level of Reactive Oxygen Species in Pollen

    PubMed Central

    Zhao, Ting Ting; Li, Fei; Jia, Xiao Na; Zhao, Xin-Ying; Zhang, Xian Sheng

    2016-01-01

    Pollen–stigma interactions are essential for pollen germination. The highly regulated process of pollen germination includes pollen adhesion, hydration, and germination on the stigma. However, the internal signaling of pollen that regulates pollen–stigma interactions is poorly understood. KINβγ is a plant-specific subunit of the SNF1-related protein kinase 1 complex which plays important roles in the regulation of plant development. Here, we showed that KINβγ was a cytoplasm- and nucleus-localized protein in the vegetative cells of pollen grains in Arabidopsis. The pollen of the Arabidopsis kinβγ mutant could not germinate on stigma, although it germinated normally in vitro. Further analysis revealed the hydration of kinβγ mutant pollen on the stigma was compromised. However, adding water to the stigma promoted the germination of the mutant pollen in vivo, suggesting that the compromised hydration of the mutant pollen led to its defective germination. In kinβγ mutant pollen, the structure of the mitochondria and peroxisomes was destroyed, and their numbers were significantly reduced compared with those in the wild type. Furthermore, we found that the kinβγ mutant exhibited reduced levels of reactive oxygen species (ROS) in pollen. The addition of H2O2 in vitro partially compensated for the reduced water absorption of the mutant pollen, and reducing ROS levels in pollen by overexpressing Arabidopsis CATALASE 3 resulted in compromised hydration of pollen on the stigma. These results indicate that Arabidopsis KINβγ is critical for the regulation of ROS levels by mediating the biogenesis of mitochondria and peroxisomes in pollen, which is required for pollen–stigma interactions during pollination. PMID:27472382

  13. Auxin Regulates Arabidopsis Anther Dehiscence, Pollen Maturation, and Filament Elongation[W

    PubMed Central

    Cecchetti, Valentina; Altamura, Maria Maddalena; Falasca, Giuseppina; Costantino, Paolo; Cardarelli, Maura

    2008-01-01

    We provide evidence on the localization, synthesis, transport, and effects of auxin on the processes occurring late in Arabidopsis thaliana stamen development: anther dehiscence, pollen maturation, and preanthesis filament elongation. Expression of auxin-sensitive reporter constructs suggests that auxin effects begin in anthers between the end of meiosis and the bilocular stage in the somatic tissues involved in the first step of dehiscence as well as in the microspores and in the junction region between anther and filament. In situ hybridizations of the auxin biosynthetic genes YUC2 and YUC6 suggest that auxin is synthesized in anthers. In agreement with the timing of auxin effects, the TIR1, AFB1, AFB2, and AFB3 auxin receptor-encoding genes are transcribed in anthers only during late stages of development starting at the end of meiosis. We found that in tir1 afb triple and quadruple mutants, anther dehiscence and pollen maturation occur earlier than in the wild type, causing the release of mature pollen grains before the completion of filament elongation. We also assessed the contribution of auxin transport to late stamen developmental processes. Our results suggest that auxin synthesized in anthers plays a major role in coordinating anther dehiscence and pollen maturation, while auxin transport contributes to the independent regulation of preanthesis filament elongation. PMID:18628351

  14. A model of plasma membrane flow and cytosis regulation in growing pollen tubes.

    PubMed

    Chavarría-Krauser, Andrés; Yejie, Du

    2011-09-21

    A model of cytosis regulation in growing pollen tubes is developed and simulations presented. The authors address the question on the minimal assumptions needed to describe the pattern of exocytosis and endocytosis reported recently by experimental biologists. Biological implications of the model are also treated. Concepts of flow and conservation of membrane material are used to pose an equation system, which describes the movement of plasma membrane in the tip of growing pollen tubes. After obtaining the central equations, relations describing the rates of endocytosis and exocytosis are proposed. Two cytosis receptors (for exocytosis and endocytosis), which have different recycling rates and activation times, suffice to describe a stable growing tube. Simulations show a very good spatial separation between endocytosis and exocytosis, in which separation is shown to depend strongly on exocytic vesicle delivery. In accordance to measurements, most vesicles in the clear zone are predicted to be endocytic. Membrane flow is essential to maintain cell polarity, and bi-directional flow seems to be a natural consequence of the proposed mechanism. For the first time, a model addressing plasma membrane flow and cytosis regulation were posed. Therefore, it represents a missing piece in an integrative model of pollen tube growth, in which cell wall mechanics, hydrodynamic fluxes and regulation mechanisms are combined. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Arabidopsis thaliana calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca(2+) concentration.

    PubMed

    Yang, Xue; Wang, Shuang-Shuang; Wang, Mei; Qiao, Zhu; Bao, Chan-Chan; Zhang, Wei

    2014-10-01

    Cytosolic free calcium ([Ca(2+)]cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca(2+)]cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K(+)] and [Ca(2+)]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca(2+)]cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.

  16. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK.

    PubMed

    Bauer, Daniela; Merz, Dale R; Pelz, Benjamin; Theisen, Kelly E; Yacyshyn, Gail; Mokranjac, Dejana; Dima, Ruxandra I; Rief, Matthias; Žoldák, Gabriel

    2015-08-18

    The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily.

  17. Nucleotides regulate the mechanical hierarchy between subdomains of the nucleotide binding domain of the Hsp70 chaperone DnaK

    PubMed Central

    Bauer, Daniela; Merz, Dale R.; Pelz, Benjamin; Theisen, Kelly E.; Yacyshyn, Gail; Mokranjac, Dejana; Dima, Ruxandra I.; Rief, Matthias; Žoldák, Gabriel

    2015-01-01

    The regulation of protein function through ligand-induced conformational changes is crucial for many signal transduction processes. The binding of a ligand alters the delicate energy balance within the protein structure, eventually leading to such conformational changes. In this study, we elucidate the energetic and mechanical changes within the subdomains of the nucleotide binding domain (NBD) of the heat shock protein of 70 kDa (Hsp70) chaperone DnaK upon nucleotide binding. In an integrated approach using single molecule optical tweezer experiments, loop insertions, and steered coarse-grained molecular simulations, we find that the C-terminal helix of the NBD is the major determinant of mechanical stability, acting as a glue between the two lobes. After helix unraveling, the relative stability of the two separated lobes is regulated by ATP/ADP binding. We find that the nucleotide stays strongly bound to lobe II, thus reversing the mechanical hierarchy between the two lobes. Our results offer general insights into the nucleotide-induced signal transduction within members of the actin/sugar kinase superfamily. PMID:26240360

  18. [PLASMALEMMAL ION TRANSPORT IN POLLEN TUBES IS REGULATED BY HYDROGEN PEROXIDE].

    PubMed

    Maksimov, N M; Breygina, M A; Yermakov, I P

    2015-01-01

    Pollen tube growth is a key step in the life cycle of seed plants, which defines the success of sexual reproduction. One of the most important contributions to this process is made by ion transport through plasmalemma, which is tightly coordinated in time and space. Different classes of signaling molecules are involved in the regulation of transmembrane ion transport including reactive oxygen species as it has been recently demonstrated. Here, using subprotoplasts isolated from pollen tubes, we have demonstrated a connection between hydrogen peroxide, on one side, and two groups of targets on the plasma membrane, on the other side: nifedipine-sensitive Ca(2+)-permeable channels and transport systems controlling membrane potential. H2O2 interaction with these targets causes the increase in cytoplasmic Ca2+ concentration and plasmalemma hyperpolarization. One of the consequences of target modification was acceleration of cell wall regeneration.

  19. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice.

    PubMed

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-07-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity.

  20. Receptor-Like Kinase RUPO Interacts with Potassium Transporters to Regulate Pollen Tube Growth and Integrity in Rice

    PubMed Central

    Liu, Lingtong; Zheng, Canhui; Kuang, Baijan; Wei, Liqin; Yan, Longfeng; Wang, Tai

    2016-01-01

    During sexual reproduction of flowering plants, the pollen tube grows fast and over a long distance within the pistil to deliver two sperms for double fertilization. Growing plant cells need to communicate constantly with external stimuli as well as monitor changes in surface tension of the cell wall and plasma membrane to coordinate these signals and internal growth machinery; however, the underlying mechanisms remain largely unknown. Here we show that the rice member of plant-specific receptor-like kinase CrRLK1Ls subfamily, Ruptured Pollen tube (RUPO), is specifically expressed in rice pollen. RUPO localizes to the apical plasma membrane and vesicle of pollen tubes and is required for male gamete transmission. K+ levels were greater in pollen of homozygous CRISPR-knockout lines than wild-type plants, and pollen tubes burst shortly after germination. We reveal the interaction of RUPO with high-affinity potassium transporters. Phosphorylation of RUPO established and dephosphorylation abolished the interaction. These results have revealed the receptor-like kinase as a regulator of high-affinity potassium transporters via phosphorylation-dependent interaction, and demonstrated a novel receptor-like kinase signaling pathway that mediates K+ homeostasis required for pollen tube growth and integrity. PMID:27447945

  1. Rac Homologues and Compartmentalized Phosphatidylinositol 4, 5-Bisphosphate Act in a Common Pathway to Regulate Polar Pollen Tube Growth

    PubMed Central

    Kost, Benedikt; Lemichez, Emmanuel; Spielhofer, Pius; Hong, Yan; Tolias, Kimberly; Carpenter, Christopher; Chua, Nam-Hai

    1999-01-01

    Pollen tube cells elongate based on actin- dependent targeted secretion at the tip. Rho family small GTPases have been implicated in the regulation of related processes in animal and yeast cells. We have functionally characterized Rac type Rho family proteins that are expressed in growing pollen tubes. Expression of dominant negative Rac inhibited pollen tube elongation, whereas expression of constitutive active Rac induced depolarized growth. Pollen tube Rac was found to accumulate at the tip plasma membrane and to physically associate with a phosphatidylinositol monophosphate kinase (PtdIns P-K) activity. Phosphatidylinositol 4, 5-bisphosphate (PtdIns 4, 5-P2), the product of PtdIns P-Ks, showed a similar intracellular localization as Rac. Expression of the pleckstrin homology (PH)-domain of phospholipase C (PLC)-δ1, which binds specifically to PtdIns 4, 5-P2, inhibited pollen tube elongation. These results indicate that Rac and PtdIns 4, 5-P2 act in a common pathway to control polar pollen tube growth and provide direct evidence for a function of PtdIns 4, 5-P2 compartmentalization in the regulation of this process. PMID:10209027

  2. Receptor-like kinases as surface regulators for RAC/ROP-mediated pollen tube growth and interaction with the pistil

    PubMed Central

    Zou, Yanjiao; Aggarwal, Mini; Zheng, Wen-Guang; Wu, Hen-Ming; Cheung, Alice Y.

    2011-01-01

    Background RAC/ROPs are RHO-type GTPases and are known to play diverse signalling roles in plants. Cytoplasmic RAC/ROPs are recruited to the cell membrane and activated in response to extracellular signals perceived and mediated by cell surface-located signalling assemblies, transducing the signals to regulate cellular processes. More than any other cell types in plants, pollen tubes depend on continuous interactions with an extracellular environment produced by their surrounding tissues as they grow within the female organ pistil to deliver sperm to the female gametophyte for fertilization. Scope We review studies on pollen tube growth that provide compelling evidence indicating that RAC/ROPs are crucial for regulating the cellular processes that underlie the polarized cell growth process. Efforts to identify cell surface regulators that mediate extracellular signals also point to RAC/ROPs being the molecular switches targeted by growth-regulating female factors for modulation to mediate pollination and fertilization. We discuss a large volume of work spanning more than two decades on a family of pollen-specific receptor kinases and some recent studies on members of the FERONIA family of receptor-like kinases (RLKs). Significance The research described shows the crucial roles that two RLK families play in transducing signals from growth regulatory factors to the RAC/ROP switch at the pollen tube apex to mediate and target pollen tube growth to the female gametophyte and signal its disintegration to achieve fertilization once inside the female chamber. PMID:22476487

  3. The Pollen Receptor Kinase LePRK2 Mediates Growth-Promoting Signals and Positively Regulates Pollen Germination and Tube Growth

    USDA-ARS?s Scientific Manuscript database

    In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato, LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here w...

  4. bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice

    PubMed Central

    Ranjan, Rajeev; Khurana, Reema; Malik, Naveen; Badoni, Saurabh; Parida, Swarup K.; Kapoor, Sanjay; Tyagi, Akhilesh K.

    2017-01-01

    Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142OEplants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes. PMID:28262713

  5. bHLH142 regulates various metabolic pathway-related genes to affect pollen development and anther dehiscence in rice.

    PubMed

    Ranjan, Rajeev; Khurana, Reema; Malik, Naveen; Badoni, Saurabh; Parida, Swarup K; Kapoor, Sanjay; Tyagi, Akhilesh K

    2017-03-06

    Apposite development of anther and its dehiscence are important for the reproductive success of the flowering plants. Recently, bHLH142, a bHLH transcription factor encoding gene of rice has been found to show anther-specific expression and mutant analyses suggest its functions in regulating tapetum differentiation and degeneration during anther development. However, our study on protein level expression and gain-of-function phenotype revealed novel aspects of its regulation and function during anther development. Temporally dissimilar pattern of bHLH142 transcript and polypeptide accumulation suggested regulation of its expression beyond transcriptional level. Overexpression of bHLH142 in transgenic rice resulted in indehiscent anthers and aborted pollen grains. Defects in septum and stomium rupture caused anther indehiscence while pollen abortion phenotype attributed to abnormal degeneration of the tapetum. Furthermore, RNA-Seq-based transcriptome analysis of tetrad and mature pollen stage anthers of wild type and bHLH142(OE)plants suggested that it might regulate carbohydrate and lipid metabolism, cell wall modification, reactive oxygen species (ROS) homeostasis and cell death-related genes during rice anther development. Thus, bHLH142 is an anther-specific gene whose expression is regulated at transcriptional and post-transcriptional/translational levels. It plays a role in pollen maturation and anther dehiscence by regulating expression of various metabolic pathways-related genes.

  6. MYB80, a regulator of tapetal and pollen development, is functionally conserved in crops.

    PubMed

    Phan, Huy A; Li, Song F; Parish, Roger W

    2012-01-01

    The Arabidopsis AtMYB80 transcription factor (formerly AtMYB103) regulate genes essential for tapetal and pollen development. One of these genes, coding for an aspartic protease (UNDEAD), may control the timing of tapetal programmed cell death (PCD). In crop plants such as rice and wheat, abiotic stresses lead to abnormal tapetal development resulting in delayed PCD. Manipulation of AtMYB80 function has been used to develop a reversible male sterility system applicable to hybrid crop production. MYB80 homologs were cloned from wheat, rice, canola and cotton. The promoters of the homologs drove temporal and spatial expression patterns of the GUS reporter gene in the tapetum and microspores of Arabidopsis anthers identical to the AtMYB80 promoter. A short region is conserved in all five MYB80 promoters. The MYB80 homolog genes, driven by the AtMYB80 or their respective promoters, rescued the atmyb80 mutant, completely restoring male fertility. The canola MYB80 was fused to the EAR (ERF-associated amphiphilic repression) repressor and canola plants transgenic for the construct exhibited premature tapetal degradation and subsequent pollen abortion. The five MYB80 homologs all shared a 44 amino acid sequence immediately adjacent to the R2R3 domain which appears to be necessary for MYB80 function.

  7. ABORTED MICROSPORES Acts as a Master Regulator of Pollen Wall Formation in Arabidopsis[C][W][OPEN

    PubMed Central

    Xu, Jie; Ding, Zhiwen; Vizcay-Barrena, Gema; Shi, Jianxin; Liang, Wanqi; Yuan, Zheng; Werck-Reichhart, Danièle; Schreiber, Lukas; Wilson, Zoe A.; Zhang, Dabing

    2014-01-01

    Mature pollen is covered by durable cell walls, principally composed of sporopollenin, an evolutionary conserved, highly resilient, but not fully characterized, biopolymer of aliphatic and aromatic components. Here, we report that ABORTED MICROSPORES (AMS) acts as a master regulator coordinating pollen wall development and sporopollenin biosynthesis in Arabidopsis thaliana. Genome-wide coexpression analysis revealed 98 candidate genes with specific expression in the anther and 70 that showed reduced expression in ams. Among these 70 members, we showed that AMS can directly regulate 23 genes implicated in callose dissociation, fatty acids elongation, formation of phenolic compounds, and lipidic transport putatively involved in sporopollenin precursor synthesis. Consistently, ams mutants showed defective microspore release, a lack of sporopollenin deposition, and a dramatic reduction in total phenolic compounds and cutin monomers. The functional importance of the AMS pathway was further demonstrated by the observation of impaired pollen wall architecture in plant lines with reduced expression of several AMS targets: the abundant pollen coat protein extracellular lipases (EXL5 and EXL6), and CYP98A8 and CYP98A9, which are enzymes required for the production of phenolic precursors. These findings demonstrate the central role of AMS in coordinating sporopollenin biosynthesis and the secretion of materials for pollen wall patterning. PMID:24781116

  8. Relationships between cystic fibrosis transmembrane conductance regulator, extracellular nucleotides and cystic fibrosis.

    PubMed

    Marcet, Brice; Boeynaems, Jean-Marie

    2006-12-01

    Cystic fibrosis (CF) is one of the most common lethal autosomal recessive genetic diseases in the Caucasian population, with a frequency of about 1 in 3000 livebirths. CF is due to a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding the CFTR protein, a cyclic adenosine 5'-monophosphate (cAMP)-regulated chloride channel localized in the apical membrane of epithelial cells. CFTR is a multifunctional protein which, in addition to be a Cl-channel, is also a regulator of multiple ion channels and other proteins. In particular CFTR has been reported to play a role in the outflow of adenosine 5'-triphosphate (ATP) from cells, but this remains controversial. Extracellular nucleotides are signaling molecules that regulate ion transport and mucociliary clearance by acting on P2 nucleotide receptors, in particular the P2Y(2) receptor. Nucleotides activating the P2Y(2) receptor represent thus one pharmacotherapeutic strategy to treat CF disease, via improvement of mucus hydration and mucociliary clearance in airways. Phase II clinical trials have recently shown that aerosolized denufosol (INS37217, Inspire(R)) improves pulmonary function in CF patients: denufosol was granted orphan drug status and phase III trials are planned. Here, we review what is known about the relationship between extracellular nucleotides and CFTR, the role of extracellular nucleotides in epithelial pathophysiology and their putative role as therapeutic agents.

  9. Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides*

    PubMed Central

    DeBerg, Hannah A.; Brzovic, Peter S.; Flynn, Galen E.; Zagotta, William N.; Stoll, Stefan

    2016-01-01

    Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel. PMID:26559974

  10. Effects of Hydrazines upon Cyclic Nucleotide Regulated Neuronal Processes

    DTIC Science & Technology

    1987-07-30

    of adenylate cyclase, however, Creutzfeldt-Jakob disease (CJD) is a slow, infec- in neuronal membranes this enzyme can be tious, progressive...61102F 2312 UPONI CYFCLIC NECLEOTIDE REGULATED NEURONAL RESP+SES 12. PORSONAIL AUTMORIS) Mark MI. Rasenick, Ph.D. 934L TYPE OF REPORT 131m. Time...CT TERMI AS Icon hN.. on powe"~ 4f mete~up *ad Idendfy by. Week mum Imr) "peLo GROUP sun. owr Neuronal Signal, Transduction, Cytoskeleton

  11. MAP18 Regulates the Direction of Pollen Tube Growth in Arabidopsis by Modulating F-Actin Organization[C][W][OA

    PubMed Central

    Zhu, Lei; Zhang, Yan; Kang, Erfang; Xu, Qiangyi; Wang, Miaoying; Rui, Yue; Liu, Baoquan; Yuan, Ming; Fu, Ying

    2013-01-01

    For fertilization to occur in plants, the pollen tube must be guided to enter the ovule via the micropyle. Previous reports have implicated actin filaments, actin binding proteins, and the tip-focused calcium gradient as key contributors to polar growth of pollen tubes; however, the regulation of directional pollen tube growth is largely unknown. We reported previously that Arabidopsis thaliana MICROTUBULE-ASSOCIATED PROTEIN18 (MAP18) contributes to directional cell growth and cortical microtubule organization. The preferential expression of MAP18 in pollen and in pollen tubes suggests that MAP18 also may function in pollen tube growth. In this study, we demonstrate that MAP18 functions in pollen tubes by influencing actin organization, rather than microtubule assembly. In vitro biochemical results indicate that MAP18 exhibits Ca2+-dependent filamentous (F)-actin-severing activity. Abnormal expression of MAP18 in map18 and MAP18 OX plants was associated with disorganization of the actin cytoskeleton in the tube apex, resulting in aberrant pollen tube growth patterns and morphologies, inaccurate micropyle targeting, and fewer fertilization events. Experiments with MAP18 mutants created by site-directed mutagenesis suggest that F-actin-severing activity is essential to the effects of MAP18 on pollen tube growth direction. Our study demonstrates that in Arabidopsis, MAP18 guides the direction of pollen tube growth by modulating actin filaments. PMID:23463774

  12. An actin-binding protein, LlLIM1, mediates calcium and hydrogen regulation of actin dynamics in pollen tubes.

    PubMed

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-08-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca(2+): LlLIM1 showed a preference for F-actin binding under low pH and low Ca(2+) concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed.

  13. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    DOE PAGES

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; ...

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allostericmore » AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.« less

  14. Structural basis of AMPK regulation by adenine nucleotides and glycogen

    SciTech Connect

    Li, Xiaodan; Wang, Lili; Zhou, X. Edward; Ke, Jiyuan; de Waal, Parker W.; Gu, Xin; Tan, M. H. Eileen; Wang, Dongye; Wu, Donghai; Xu, H. Eric; Melcher, Karsten

    2014-11-21

    AMP-activated protein kinase (AMPK) is a central cellular energy sensor and regulator of energy homeostasis, and a promising drug target for the treatment of diabetes, obesity, and cancer. Here we present low-resolution crystal structures of the human α1β2γ1 holo-AMPK complex bound to its allosteric modulators AMP and the glycogen-mimic cyclodextrin, both in the phosphorylated (4.05 Å) and non-phosphorylated (4.60 Å) state. In addition, we have solved a 2.95 Å structure of the human kinase domain (KD) bound to the adjacent autoinhibitory domain (AID) and have performed extensive biochemical and mutational studies. Altogether, these studies illustrate an underlying mechanism of allosteric AMPK modulation by AMP and glycogen, whose binding changes the equilibria between alternate AID (AMP) and carbohydrate-binding module (glycogen) interactions.

  15. Bumble bees regulate their intake of essential protein and lipid pollen macronutrients.

    PubMed

    Vaudo, A D; Stabler, D; Patch, H M; Tooker, J F; Grozinger, C M; Wright, G A

    2016-12-15

    Bee population declines are linked to the reduction of nutritional resources due to land-use intensification, yet we know little about the specific nutritional needs of many bee species. Pollen provides bees with their primary source of protein and lipids, but nutritional quality varies widely among host-plant species. Therefore, bees might have adapted to assess resource quality and adjust their foraging behavior to balance nutrition from multiple food sources. We tested the ability of two bumble bee species, Bombus terrestris and Bombus impatiens, to regulate protein and lipid intake. We restricted B. terrestris adults to single synthetic diets varying in protein:lipid ratios (P:L). The bees over-ate protein on low-fat diets and over-ate lipid on high-fat diets to reach their targets of lipid and protein, respectively. The bees survived best on a 10:1 P:L diet; the risk of dying increased as a function of dietary lipid when bees ate diets with lipid contents greater than 5:1 P:L. Hypothesizing that the P:L intake target of adult worker bumble bees was between 25:1 and 5:1, we presented workers from both species with unbalanced but complementary paired diets to determine whether they self-select their diet to reach a specific intake target. Bees consumed similar amounts of proteins and lipids in each treatment and averaged a 14:1 P:L for B. terrestris and 12:1 P:L for B. impatiens These results demonstrate that adult worker bumble bees likely select foods that provide them with a specific ratio of P:L. These P:L intake targets could affect pollen foraging in the field and help explain patterns of host-plant species choice by bumble bees.

  16. Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development.

    PubMed

    Cheng, Jintao; Wang, Zhenyu; Yao, Fengzhen; Gao, Lihong; Ma, Si; Sui, Xiaolei; Zhang, Zhenxian

    2015-06-01

    Efficient sugar transport is needed to support the high metabolic activity of pollen tubes as they grow through the pistil. Failure of transport results in male sterility. Although sucrose transporters have been shown to play a role in pollen tube development, the role of hexoses and hexose transporters is not as well established. The pollen of some species can grow in vitro on hexose as well as on sucrose, but knockouts of individual hexose transporters have not been shown to impair fertilization, possibly due to transporter redundancy. Here, the functions of CsHT1, a hexose transporter from cucumber (Cucumis sativus), are studied using a combination of heterologous expression in yeast (Saccharomyces cerevisiae), histochemical and immunohistochemical localization, and reverse genetics. The results indicate that CsHT1 is a plasma membrane-localized hexose transporter with high affinity for glucose, exclusively transcribed in pollen development and expressed both at the levels of transcription and translation during pollen grain germination and pollen tube growth. Overexpression of CsHT1 in cucumber pollen results in a higher pollen germination ratio and longer pollen tube growth than wild-type pollen in glucose- or galactose-containing medium. By contrast, antisense suppression of CsHT1 leads to inhibition of pollen germination and pollen tube elongation in the same medium and results in a decrease of seed number per fruit and seed size when antisense transgenic pollen is used to fertilize wild-type or transgenic cucumber plants. The important role of CsHT1 in pollen germination, pollen tube growth, and seed development is discussed.

  17. Down-Regulating CsHT1, a Cucumber Pollen-Specific Hexose Transporter, Inhibits Pollen Germination, Tube Growth, and Seed Development1[OPEN

    PubMed Central

    Cheng, Jintao; Wang, Zhenyu; Yao, Fengzhen; Gao, Lihong; Ma, Si; Zhang, Zhenxian

    2015-01-01

    Efficient sugar transport is needed to support the high metabolic activity of pollen tubes as they grow through the pistil. Failure of transport results in male sterility. Although sucrose transporters have been shown to play a role in pollen tube development, the role of hexoses and hexose transporters is not as well established. The pollen of some species can grow in vitro on hexose as well as on sucrose, but knockouts of individual hexose transporters have not been shown to impair fertilization, possibly due to transporter redundancy. Here, the functions of CsHT1, a hexose transporter from cucumber (Cucumis sativus), are studied using a combination of heterologous expression in yeast (Saccharomyces cerevisiae), histochemical and immunohistochemical localization, and reverse genetics. The results indicate that CsHT1 is a plasma membrane-localized hexose transporter with high affinity for glucose, exclusively transcribed in pollen development and expressed both at the levels of transcription and translation during pollen grain germination and pollen tube growth. Overexpression of CsHT1 in cucumber pollen results in a higher pollen germination ratio and longer pollen tube growth than wild-type pollen in glucose- or galactose-containing medium. By contrast, antisense suppression of CsHT1 leads to inhibition of pollen germination and pollen tube elongation in the same medium and results in a decrease of seed number per fruit and seed size when antisense transgenic pollen is used to fertilize wild-type or transgenic cucumber plants. The important role of CsHT1 in pollen germination, pollen tube growth, and seed development is discussed. PMID:25888616

  18. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides

    PubMed Central

    Lehninger, Albert L.; Vercesi, Anibal; Bababunmi, Enitan A.

    1978-01-01

    Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca2+ and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca2+ is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as β-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca2+. Successive cycles of Ca2+ release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca2+-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca2+, mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport. Images PMID:25436

  19. Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.

    PubMed

    Lehninger, A L; Vercesi, A; Bababunmi, E A

    1978-04-01

    Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca(2+) and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca(2+) is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as beta-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca(2+). Successive cycles of Ca(2+) release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca(2+)-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca(2+), mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport.

  20. The Regulation of Vesicle Trafficking by Small GTPases and Phospholipids during Pollen Tube Growth

    USDA-ARS?s Scientific Manuscript database

    Polarized and directional growth of pollen tubes is the only means by which immotile sperm of flowering plants reach the deeply embedded female gametes for fertilization. Vesicle trafficking is among the most critical cellular activities for pollen tube growth. Vesicle trafficking maintains membrane...

  1. Cytosolic nucleotides block and regulate the Arabidopsis vacuolar anion channel AtALMT9.

    PubMed

    Zhang, Jingbo; Martinoia, Enrico; De Angeli, Alexis

    2014-09-12

    The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.

  2. Using pollen grains as novel hydrophilic solid-phase extraction sorbents for the simultaneous determination of 16 plant growth regulators.

    PubMed

    Lu, Qian; Wu, Jian-Hong; Yu, Qiong-Wei; Feng, Yu-Qi

    2014-11-07

    In this article, pollen grains were for the first time used as a hydrophilic solid-phase extraction (HILIC-SPE) sorbent for the determination of 16 plant growth regulators (PGRs) in fruits and vegetables. Fourier transform infrared spectroscopy (FT-IR), scanning electronic microscopy (SEM) and nitrogen sorption porosimetry (NSP) were used to investigate the chemical structure and the surface properties of the pollen grains. Pollen grains exhibited an excellent adsorption capacity for some polar compounds due to their particular functional groups. Several parameters influencing extraction performance were investigated. A green and simple HILIC-SPE-method using pollen grain cartridge for purification of fruit and vegetable extractions, followed by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS/MS) was established. Good linear relationships were obtained for 16 PGRs with correlation coefficients (R) above 0.9980. The limits of detection (LODs) of 16 PGRs in cucumber were in the range of 0.01-1.10 μg · kg(-1). Reproducibility of the method was evaluated by intra-day and inter-day precisions with relative standard deviations (RSDs), which were less than 14.4%. We successfully applied this methodology to analyze 16 PGRs in 8 different kinds of fruits and vegetables. The recoveries from samples spiked with 16 PGRs were from 80.5% to 119.2%, with relative standard deviations less than 15.0%.

  3. Inositol polyphosphate 5-phosphatase-controlled Ins(1,4,5)P3/Ca2+ is crucial for maintaining pollen dormancy and regulating early germination of pollen.

    PubMed

    Wang, Yuan; Chu, Yu-Jia; Xue, Hong-Wei

    2012-06-01

    Appropriate pollen germination is crucial for plant reproduction. Previous studies have revealed the importance of dehydration in maintaining pollen dormancy; here, we show that phosphatidylinositol pathway-controlled Ins(1,4,5)P(3)/Ca(2+) levels are crucial for maintaining pollen dormancy in Arabidopsis thaliana. An interesting phenotype, precocious pollen germination within anthers, results from a disruption of inositol polyphosphate 5-phosphatase 12 (5PT12). The knockout mutant 5pt12 has normal early pollen development and pollen dehydration, and exhibits hypersensitive ABA responses, indicating that precocious pollen germination is not caused either by abnormal dehydration or by suppressed ABA signaling. Deficiency of 5PT13 (a close paralog of 5PT12) synergistically enhances precocious pollen germination. Both basal Ins(1,4,5)P(3) levels and endogenous Ca(2+) levels are elevated in pollen from 5pt12 mutants, and 5pt12 5pt13 double mutants show an even higher precocious germination rate along with much higher levels of Ins(1,4,5)P(3)/Ca(2+). Strikingly, exogenous Ca(2+) stimulates the germination of wild-type pollen at floral stage 12, even in very low humidity, both in vitro and in vivo, and treatment with BAPTA, a [Ca(2+)](cyt) inhibitor, reduces the precocious pollen germination rates of 5pt12, 5pt13 and 5pt12 5pt13 mutants. These results indicate that the increase in the levels of Ins(1,4,5)P(3)/Ca(2+) caused by deficiency of inositol polyphosphate 5-phosphatases is sufficient to break pollen dormancy and to trigger early germination. The study reveals that independent of dehydration, the control of Ins(1,4,5)P(3)/Ca(2+) levels by Inositol polyphosphate 5-phosphatases is crucial for maintaining pollen dormancy.

  4. Regulation of IMP dehydrogenase gene expression by its end products, guanine nucleotides.

    PubMed Central

    Glesne, D A; Collart, F R; Huberman, E

    1991-01-01

    To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of cellular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time- and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides. Images PMID:1717828

  5. Cloning and characterisation of a putative pollen-specific polygalacturonase gene (CpPG1) differentially regulated during pollen development in zucchini (Cucurbita pepo L.).

    PubMed

    Carvajal, F; Garrido, D; Jamilena, M; Rosales, R

    2014-03-01

    Studies in zucchini (Cucurbita pepo L. spp. pepo) pollen have been limited to the viability and morphology of the mature pollen grain. The enzyme polygalacturonase (PG) is involved in pollen development and pollination in many species. In this work, we study anther and pollen development of C. pepo and present the cloning and characterisation of a putative PG CpPG1 (Accession no. HQ232488) from pollen cDNA in C. pepo. The predicted protein for CpPG1 has 416 amino acids, with a high homology to other pollen PGs, such as P22 from Oenothera organensis (76%) and PGA3 from Arabidopsis thaliana (73%). CpPG1 belongs to clade C, which comprises PGs expressed in pollen, and presents a 34 amino acid signal peptide for secretion towards the cell wall. DNA-blot analysis revealed that there are at least another two genes that code for PGs in C. pepo. The spatial and temporal accumulation of CpPG1 was studied by semi-quantitative- and qRT-PCR. In addition, mRNA was detected only in anthers, pollen and the rudimentary anthers of bisexual flowers (only present in some zucchini cultivars under certain environmental conditions that trigger anther development in the third whorl of female flowers). However, no expression was detected in cotyledons, stem or fruit. Furthermore, CpPG1 mRNA was accumulated throughout anther development, with the highest expression found in mature pollen. Similarly, exo-PG activity increased from immature anther stages to mature anthers and mature pollen. Overall, these data support the pollen specificity of this gene and suggest an involvement of CpPG1 in pollen development in C. pepo. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

  6. Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene.

    PubMed Central

    van Tunen, A J; Mur, L A; Brouns, G S; Rienstra, J D; Koes, R E; Mol, J N

    1990-01-01

    We have analyzed the spatial and temporal activities of chalcone flavanone isomerase (chi) A and B gene promoters from petunia. To study the tandem promoter regulation of chiA, various chiA promoter fragments were fused with the beta-glucuronidase (GUS) reporter gene. Analysis of transgenic plants containing these chimeric genes provided definitive proof that the chiA coding region is regulated by two distinct promoters (designated PA1 and PA2). We also showed that both promoters can function independently and that the chiA PA1 promoter is expressed in limb (epidermal and parenchyma cells), tube (inner epidermal and parenchyma cells), seed (seed coat, endosperm, and embryo), sepal, leaf, and stem. The use of chiA and chiB promoters in the regulation of anther- and pollen-specific gene expression has been studied. By analyzing transgenic plants containing chimeric genes consisting of chiA and B promoter fragments and the GUS reporter gene, we were able to identify a 0.44-kilobase chiA PA2 promoter fragment that drives pollen-specific gene expression and a 1.75-kilobase chiB PB promoter fragment that confers anther-specific (pollen and tapetum cells) expression to the GUS gene. PMID:2152165

  7. Bee Pollen

    MedlinePlus

    ... nectar and bee saliva. Pollens come from many plants, so the contents of bee pollen can vary ... joint pain (rheumatism), painful urination, prostate conditions, and radiation ... or other ingredients in bee pollen are effective as treatment.

  8. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    PubMed

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis.

  9. Viral Polymerase-Helicase Complexes Regulate Replication Fidelity To Overcome Intracellular Nucleotide Depletion

    PubMed Central

    Stapleford, Kenneth A.; Rozen-Gagnon, Kathryn; Das, Pratyush Kumar; Saul, Sirle; Poirier, Enzo Z.; Blanc, Hervé; Vidalain, Pierre-Olivier; Merits, Andres

    2015-01-01

    ABSTRACT To date, the majority of work on RNA virus replication fidelity has focused on the viral RNA polymerase, while the potential role of other viral replicase proteins in this process is poorly understood. Previous studies used resistance to broad-spectrum RNA mutagens, such as ribavirin, to identify polymerases with increased fidelity that avoid misincorporation of such base analogues. We identified a novel variant in the alphavirus viral helicase/protease, nonstructural protein 2 (nsP2) that operates in concert with the viral polymerase nsP4 to further alter replication complex fidelity, a functional linkage that was conserved among the alphavirus genus. Purified chikungunya virus nsP2 presented delayed helicase activity of the high-fidelity enzyme, and yet purified replication complexes manifested stronger RNA polymerization kinetics. Because mutagenic nucleoside analogs such as ribavirin also affect intracellular nucleotide pools, we addressed the link between nucleotide depletion and replication fidelity by using purine and pyrimidine biosynthesis inhibitors. High-fidelity viruses were more resistant to these conditions, and viral growth could be rescued by the addition of exogenous nucleosides, suggesting that mutagenesis by base analogues requires nucleotide pool depletion. This study describes a novel function for nsP2, highlighting the role of other components of the replication complex in regulating viral replication fidelity, and suggests that viruses can alter their replication complex fidelity to overcome intracellular nucleotide-depleting conditions. IMPORTANCE Previous studies using the RNA mutagen ribavirin to select for drug-resistant variants have highlighted the essential role of the viral RNA-dependent RNA polymerase in regulating replication fidelity. However, the role of other viral replicase components in replication fidelity has not been studied in detail. We identified here an RNA mutagen-resistant variant of the nsP2 helicase

  10. A Fluorometric Activity Assay for Light-Regulated Cyclic-Nucleotide-Monophosphate Actuators.

    PubMed

    Schumacher, Charlotte Helene; Körschen, Heinz G; Nicol, Christopher; Gasser, Carlos; Seifert, Reinhard; Schwärzel, Martin; Möglich, Andreas

    2016-01-01

    As a transformative approach in neuroscience and cell biology, optogenetics grants control over manifold cellular events with unprecedented spatiotemporal definition, reversibility, and noninvasiveness. Sensory photoreceptors serve as genetically encoded, light-regulated actuators and hence embody the cornerstone of optogenetics. To expand the scope of optogenetics, ever more naturally occurring photoreceptors are being characterized, and synthetic photoreceptors with customized, light-regulated function are being engineered. Perturbational control over intracellular cyclic-nucleotide-monophosphate (cNMP) levels is achieved via sensory photoreceptors that catalyze the making and breaking of these second messengers in response to light. To facilitate discovery, engineering and quantitative characterization of such light-regulated cNMP actuators, we have developed an efficient fluorometric assay. Both the formation and the hydrolysis of cNMPs are accompanied by proton release which can be quantified with the fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). This assay equally applies to nucleotide cyclases, e.g., blue-light-activated bPAC, and to cNMP phosphodiesterases, e.g., red-light-activated LAPD. Key benefits include potential for parallelization and automation, as well as suitability for both purified enzymes and crude cell lysates. The BCECF assay hence stands to accelerate discovery and characterization of light-regulated actuators of cNMP metabolism.

  11. Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene.

    PubMed

    Francis, Kirk E; Lam, Sandy Y; Copenhaver, Gregory P

    2006-11-01

    Arabidopsis (Arabidopsis thaliana) QUARTET (QRT) genes are required for pollen separation during normal floral development. In qrt mutants, the four products of microsporogenesis remain fused and pollen grains are released as tetrads. In Arabidopsis, tetrad analysis in qrt mutants has been used to map all five centromeres, easily distinguish sporophytic from gametophytic mutations, and accurately assess crossover interference. Using a combination of forward and reverse genetics, we have identified the gene responsible for the qrt1 phenotype. Annotation predicts that QRT1 encodes a pectin methylesterase (PME), and enzymatic assays of QRT1 expressed in Escherichia coli indicate that QRT1 has PME activity. Promoter and transcription analysis demonstrate QRT1 is expressed in anther tissues shortly after meiosis is complete. Unexpectedly, the QRT1 promoter is also active in a variety of developmentally unrelated tissues, including developing guard cells, the hypocotyl-root transition zone, areas of lateral root emergence, and floral nectaries. PMEs constitute a large gene family in Arabidopsis, are involved in cell wall loosening, and have been implicated in various aspects of floral development and pollen tube elongation. The identification of QRT1 as a PME contributes to our understanding of pollen development and may help to provide valuable genetic tools in other plant species.

  12. Nucleotide levels regulate germline proliferation through modulating GLP-1/Notch signaling in C. elegans

    PubMed Central

    Chi, Congwu; Ronai, Diana; Than, Minh T.; Walker, Cierra J.; Sewell, Aileen K.; Han, Min

    2016-01-01

    Animals alter their reproductive programs to accommodate changes in nutrient availability, yet the connections between known nutrient-sensing systems and reproductive programs are underexplored, and whether there is a mechanism that senses nucleotide levels to coordinate germline proliferation is unknown. We established a model system in which nucleotide metabolism is perturbed in both the nematode Caenorhabditis elegans (cytidine deaminases) and its food (Escherichia coli); when fed food with a low uridine/thymidine (U/T) level, germline proliferation is arrested. We provide evidence that this impact of U/T level on the germline is critically mediated by GLP-1/Notch and MPK-1/MAPK, known to regulate germline mitotic proliferation. This germline defect is suppressed by hyperactivation of glp-1 or disruption of genes downstream from glp-1 to promote meiosis but not by activation of the IIS or TORC1 pathways. Moreover, GLP-1 expression is post-transcriptionally modulated by U/T levels. Our results reveal a previously unknown nucleotide-sensing mechanism for controlling reproductivity. PMID:26833730

  13. Structure of nucleotide-binding domain 1 of the cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Lewis, Hal A; Buchanan, Sean G; Burley, Stephen K; Conners, Kris; Dickey, Mark; Dorwart, Michael; Fowler, Richard; Gao, Xia; Guggino, William B; Hendrickson, Wayne A; Hunt, John F; Kearins, Margaret C; Lorimer, Don; Maloney, Peter C; Post, Kai W; Rajashankar, Kanagalaghatta R; Rutter, Marc E; Sauder, J Michael; Shriver, Stephanie; Thibodeau, Patrick H; Thomas, Philip J; Zhang, Marie; Zhao, Xun; Emtage, Spencer

    2004-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1–NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments. PMID:14685259

  14. Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

    PubMed Central

    Ljungdahl, Per O.; Daignan-Fornier, Bertrand

    2012-01-01

    Ever since the beginning of biochemical analysis, yeast has been a pioneering model for studying the regulation of eukaryotic metabolism. During the last three decades, the combination of powerful yeast genetics and genome-wide approaches has led to a more integrated view of metabolic regulation. Multiple layers of regulation, from suprapathway control to individual gene responses, have been discovered. Constitutive and dedicated systems that are critical in sensing of the intra- and extracellular environment have been identified, and there is a growing awareness of their involvement in the highly regulated intracellular compartmentalization of proteins and metabolites. This review focuses on recent developments in the field of amino acid, nucleotide, and phosphate metabolism and provides illustrative examples of how yeast cells combine a variety of mechanisms to achieve coordinated regulation of multiple metabolic pathways. Importantly, common schemes have emerged, which reveal mechanisms conserved among various pathways, such as those involved in metabolite sensing and transcriptional regulation by noncoding RNAs or by metabolic intermediates. Thanks to the remarkable sophistication offered by the yeast experimental system, a picture of the intimate connections between the metabolomic and the transcriptome is becoming clear. PMID:22419079

  15. Sounds of silence: synonymous nucleotides as a key to biological regulation and complexity

    PubMed Central

    Shabalina, Svetlana A.; Spiridonov, Nikolay A.; Kashina, Anna

    2013-01-01

    Messenger RNA is a key component of an intricate regulatory network of its own. It accommodates numerous nucleotide signals that overlap protein coding sequences and are responsible for multiple levels of regulation and generation of biological complexity. A wealth of structural and regulatory information, which mRNA carries in addition to the encoded amino acid sequence, raises the question of how these signals and overlapping codes are delineated along non-synonymous and synonymous positions in protein coding regions, especially in eukaryotes. Silent or synonymous codon positions, which do not determine amino acid sequences of the encoded proteins, define mRNA secondary structure and stability and affect the rate of translation, folding and post-translational modifications of nascent polypeptides. The RNA level selection is acting on synonymous sites in both prokaryotes and eukaryotes and is more common than previously thought. Selection pressure on the coding gene regions follows three-nucleotide periodic pattern of nucleotide base-pairing in mRNA, which is imposed by the genetic code. Synonymous positions of the coding regions have a higher level of hybridization potential relative to non-synonymous positions, and are multifunctional in their regulatory and structural roles. Recent experimental evidence and analysis of mRNA structure and interspecies conservation suggest that there is an evolutionary tradeoff between selective pressure acting at the RNA and protein levels. Here we provide a comprehensive overview of the studies that define the role of silent positions in regulating RNA structure and processing that exert downstream effects on proteins and their functions. PMID:23293005

  16. Regulation of cytochrome c oxidase by adenylic nucleotides. Is oxidative phosphorylation feedback regulated by its end-products?

    PubMed

    Beauvoit, B; Rigoulet, M

    2001-01-01

    Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria. The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force. However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides. Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane. These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency. Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations. In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis. Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria. Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships. The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism.

  17. Human Rho Guanine Nucleotide Exchange Factor 11 (ARHGEF11) Regulates Dendritic Morphogenesis

    PubMed Central

    Mizuki, Yutaka; Takaki, Manabu; Sakamoto, Shinji; Okamoto, Sojiro; Kishimoto, Makiko; Okahisa, Yuko; Itoh, Masahiko; Yamada, Norihito

    2016-01-01

    Disturbances of synaptic connectivity during perinatal and adolescent periods have been hypothesized to be related to the pathophysiology of schizophrenia. Rho guanine nucleotide exchange factor 11 (ARHGEF11) is a specific guanine nucleotide exchange factors (GEF) for RhoA, which is a critical regulator of actin cytoskeleton dynamics and organization of dendritic spines and inhibitor of spine maintenance. ARHGEF11 variants are reported to be associated with a higher risk for the onset of schizophrenia in a Japanese population; however, how ARHGEF11 contributes to the pathogenesis of schizophrenia in dendritic spines is unknown. Therefore, we first studied the distribution, binding, and function of ARHGEF11 in the dendritic spines of the rat cerebral cortex. After subcellular fractionation of the rat cerebral cortex, ARHGEF11 was detected with synaptophysin and post-synaptic density protein 95 (PSD-95) in the P2 fractions including synaptosomal fractions containing presynaptic and postsynaptic density proteins. Endogenous ARHGEF11 was coimmunoprecipitated with synaptophysin or PSD-95. In cortical primary neurons at 28 days in vitro, immunostaining revealed that ARHGEF11 located in the dendrites and dendritic spines and colocalized with PSD-95 and synaptophysin. Overexpression of exogenous ARHGEF11 significantly decreased the number of spines (p = 0.008). These results indicate that ARHGEF11 is likely to be associated with synaptic membranes and regulation of spine. PMID:28036092

  18. MONENSIN SENSITIVITY1 (MON1)/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-Mediated Rab7 Activation Regulates Tapetal Programmed Cell Death and Pollen Development1[OPEN

    PubMed Central

    Wong, Wing Shing; Wang, Xiangfeng; Gao, Caiji; Ueda, Takashi; Jiang, Liwen

    2017-01-01

    Programmed cell death (PCD)-triggered degradation of plant tapetum is essential for microspore development and pollen coat formation; however, little is known about the cellular mechanism regulating tapetal PCD. Here, we demonstrate that Rab7-mediated vacuolar transport of tapetum degradation-related cysteine proteases is crucial for tapetal PCD and pollen development in Arabidopsis (Arabidopsis thaliana), with the following evidence: (1) The monensin sensitivity1 (mon1) mutants, which are defective in Rab7 activation, showed impaired male fertility due to a combined defect in both tapetum and male gametophyte development. (2) In anthers, MON1 showed preferential high level expression in tapetal cell layers and pollen. (3) The mon1 mutants exhibited delayed tapetum degeneration and tapetal PCD, resulting in abnormal pollen coat formation and decreased male fertility. (4) MON1/CALCIUM CAFFEINE ZINC SENSITIVITY1 (CCZ1)-mediated Rab7 activation was indispensable for vacuolar trafficking of tapetum degradation-related cysteine proteases, supporting that PCD-triggered tapetum degeneration requires Rab7-mediated vacuolar trafficking of these cysteine proteases. (5) MON1 mutations also resulted in defective pollen germination and tube growth. Taken together, tapetal PCD and pollen development require successful MON1/CCZ1-mediated vacuolar transport in Arabidopsis. PMID:27799422

  19. Dissecting Enzyme Regulation by Multiple Allosteric Effectors: Nucleotide Regulation of Aspartate Transcarbamoylase†

    PubMed Central

    Rabinowitz, Joshua D.; Hsiao, Jennifer J.; Gryncel, Kimberly R.; Kantrowitz, Evan R.; Feng, Xiao-Jiang; Li, Genyuan; Rabitz, Herschel

    2009-01-01

    The enzyme aspartate transcarbamoylase (ATCase, EC 2.1.3.2 of Escherichia coli), which catalyzes the committed step of pyrimidine biosynthesis, is allosterically regulated by all four ribonucleoside triphosphates (NTPs) in a nonlinear manner. Here, we dissect this regulation using the recently developed approach of random sampling–high-dimensional model representation (RS–HDMR). ATCase activity was measured in vitro at 300 random NTP concentration combinations, each involving (consistent with in vivo conditions) all four NTPs being present. These data were then used to derive a RS–HDMR model of ATCase activity over the full four-dimensional NTP space. The model accounted for 90% of the variance in the experimental data. Its main elements were positive ATCase regulation by ATP and negative by CTP, with the negative effects of CTP dominating the positive ones of ATP when both regulators were abundant (i.e., a negative cooperative effect of ATP × CTP). Strong sensitivity to both ATP and CTP concentrations occurred in their physiological concentration ranges. UTP had only a slight effect, and GTP had almost none. These findings support a predominant role of CTP and ATP in ATCase regulation. The general approach provides a new paradigm for dissecting multifactorial regulation of biological molecules and processes. PMID:18454556

  20. Hypoxia-inducible factor-1α regulates the expression of nucleotide excision repair proteins in keratinocytes

    PubMed Central

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L.; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R.; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1α (HIF-1α) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1α increased XPC mRNA expression due to competition between HIF-1α and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1α protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1α also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1α is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1α downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1α in the repair of UVB-induced DNA damage. PMID:19934262

  1. Hypoxia-inducible factor-1alpha regulates the expression of nucleotide excision repair proteins in keratinocytes.

    PubMed

    Rezvani, Hamid Reza; Mahfouf, Walid; Ali, Nsrein; Chemin, Cecile; Ged, Cecile; Kim, Arianna L; de Verneuil, Hubert; Taïeb, Alain; Bickers, David R; Mazurier, Frédéric

    2010-01-01

    The regulation of DNA repair enzymes is crucial for cancer prevention, initiation, and therapy. We have studied the effect of ultraviolet B (UVB) radiation on the expression of the two nucleotide excision repair factors (XPC and XPD) in human keratinocytes. We show that hypoxia-inducible factor-1alpha (HIF-1alpha) is involved in the regulation of XPC and XPD. Early UVB-induced downregulation of HIF-1alpha increased XPC mRNA expression due to competition between HIF-1alpha and Sp1 for their overlapping binding sites. Late UVB-induced enhanced phosphorylation of HIF-1alpha protein upregulated XPC mRNA expression by direct binding to a separate hypoxia response element (HRE) in the XPC promoter region. HIF-1alpha also regulated XPD expression by binding to a region of seven overlapping HREs in its promoter. Quantitative chromatin immunoprecipitation assays further revealed putative HREs in the genes encoding other DNA repair proteins (XPB, XPG, CSA and CSB), suggesting that HIF-1alpha is a key regulator of the DNA repair machinery. Analysis of the repair kinetics of 6-4 photoproducts and cyclobutane pyrimidine dimers also revealed that HIF-1alpha downregulation led to an increased rate of immediate removal of both photolesions but attenuated their late removal following UVB irradiation, indicating the functional effects of HIF-1alpha in the repair of UVB-induced DNA damage.

  2. Determinants of ligand selectivity in a cyclic nucleotide-regulated potassium channel.

    PubMed

    Pessoa, João; Fonseca, Fátima; Furini, Simone; Morais-Cabral, João H

    2014-07-01

    Cyclic nucleotide-binding (CNB) domains regulate the activity of channels, kinases, exchange factors, and transcription factors. These proteins are highly variable in their ligand selectivity; some are highly selective for either cAMP or cGMP, whereas others are not. Several molecular determinants of ligand selectivity in CNB domains have been defined, but these do not provide a complete view of the selectivity mechanism. We performed a thorough analysis of the ligand-binding properties of mutants of the CNB domain from the MlotiK1 potassium channel. In particular, we defined which residues specifically favor cGMP or cAMP. Inversion of ligand selectivity, from favoring cAMP to favoring cGMP, was only achieved through a combination of three mutations in the ligand-binding pocket. We determined the x-ray structure of the triple mutant bound to cGMP and performed molecular dynamics simulations and a biochemical analysis of the effect of the mutations. We concluded that the increase in cGMP affinity and selectivity does not result simply from direct interactions between the nucleotide base and the amino acids introduced in the ligand-binding pocket residues. Rather, tighter cGMP binding over cAMP results from the polar chemical character of the mutations, from greater accessibility of water molecules to the ligand in the bound state, and from an increase in the structural flexibility of the mutated binding pocket. © 2014 Pessoa et al.

  3. Analysis of a cloned colicin Ib gene: complete nucleotide sequence and implications for regulation of expression.

    PubMed Central

    Varley, J M; Boulnois, G J

    1984-01-01

    The complete nucleotide sequence of a 2,971 base pair EcoRI fragment carrying the structural gene for colicin Ib has been determined. The length of the gene is 1,881 nucleotides which is predicted to produce a protein of 626 amino acids and of molecular weight 71,364. The structural gene is flanked by likely promoter and terminator signals and in between the promoter and the ribosome binding site is an inverted repeat sequence which resembles other sequences known to bind the LexA protein. Further analysis of the 5' flanking sequences revealed a second region which may act either as a second LexA binding site and/or in the binding of cyclic AMP receptor protein. Comparison of the predicted amino acid sequence of colicin Ib with that of colicins A and E1 reveals localised homology. The implications of these similarities in the proteins and of regulation of the colicin Ib structural gene are discussed. Images PMID:6091036

  4. Conformational dynamics of a G-protein α subunit is tightly regulated by nucleotide binding

    PubMed Central

    Goricanec, David; Stehle, Ralf; Egloff, Pascal; Grigoriu, Simina; Wagner, Gerhard; Hagn, Franz

    2016-01-01

    Heterotrimeric G proteins play a pivotal role in the signal-transduction pathways initiated by G-protein–coupled receptor (GPCR) activation. Agonist–receptor binding causes GDP-to-GTP exchange and dissociation of the Gα subunit from the heterotrimeric G protein, leading to downstream signaling. Here, we studied the internal mobility of a G-protein α subunit in its apo and nucleotide-bound forms and characterized their dynamical features at multiple time scales using solution NMR, small-angle X-ray scattering, and molecular dynamics simulations. We find that binding of GTP analogs leads to a rigid and closed arrangement of the Gα subdomain, whereas the apo and GDP-bound forms are considerably more open and dynamic. Furthermore, we were able to detect two conformational states of the Gα Ras domain in slow exchange whose populations are regulated by binding to nucleotides and a GPCR. One of these conformational states, the open state, binds to the GPCR; the second conformation, the closed state, shows no interaction with the receptor. Binding to the GPCR stabilizes the open state. This study provides an in-depth analysis of the conformational landscape and the switching function of a G-protein α subunit and the influence of a GPCR in that landscape. PMID:27298341

  5. Four closely-related RING-type E3 ligases, APD1-4, are involved in pollen mitosis II regulation in Arabidopsis.

    PubMed

    Luo, Guo; Gu, Hongya; Liu, Jingjing; Qu, Li-Jia

    2012-10-01

    Ubiquitination of proteins is one of the critical regulatory mechanisms in eukaryotes. In higher plants, protein ubiquitination plays an essential role in many biological processes, including hormone signaling, photomorphogenesis, and pathogen defense. However, the roles of protein ubiquitination in the reproductive process are not clear. In this study, we identified four plant-specific RING-finger genes designated Aberrant Pollen Development 1 (APD1) to APD4, as regulators of pollen mitosis II (PMII) in Arabidopsis thaliana (L.). The apd1 apd2 double mutant showed a significantly increased percentage of bicellular-like pollen at the mature pollen stage. Further downregulation of the APD3 and APD4 transcripts in apd1 apd2 by RNA interference (RNAi) resulted in more severe abnormal bicellular-like pollen phenotypes than in apd1 apd2, suggesting that cell division was defective in male gametogenesis. All of the four genes were expressed in multiple stages at different levels during male gametophyte development. Confocal analysis using green florescence fusion proteins (GFP) GFP-APD1 and GFP-APD2 showed that APDs are associated with intracellular membranes. Furthermore, APD2 had E2-dependent E3 ligase activity in vitro, and five APD2-interacting proteins were identified. Our results suggest that these four genes may be involved, redundantly, in regulating the PMII process during male gametogenesis. © 2012 Institute of Botany, Chinese Academy of Sciences.

  6. Antagonistic regulation of native Ca2+- and ATP-sensitive cation channels in brain capillaries by nucleotides and decavanadate.

    PubMed

    Csanády, László; Adam-Vizi, Vera

    2004-06-01

    Regulation by cytosolic nucleotides of Ca2+- and ATP-sensitive nonselective cation channels (CA-NSCs) in rat brain capillary endothelial cells was studied in excised inside-out patches. Open probability (Po) was suppressed by cytosolic nucleotides with apparent KI values of 17, 9, and 2 microM for ATP, ADP, and AMP, as a consequence of high-affinity inhibition of channel opening rate and low-affinity stimulation of closing rate. Cytosolic [Ca2+] and voltage affected inhibition of Po, but not of opening rate, by ATP, suggesting that the conformation of the nucleotide binding site is influenced only by the state of the channel gate, not by that of the Ca2+ and voltage sensors. ATP inhibition was unaltered by channel rundown. Nucleotide structure affected inhibitory potency that was little sensitive to base substitutions, but was greatly diminished by 3'-5' cyclization, removal of all phosphates, or complete omission of the base. In contrast, decavanadate potently (K1/2 = 90 nM) and robustly stimulated Po, and functionally competed with inhibitory nucleotides. From kinetic analyses we conclude that (a) ATP, ADP, and AMP bind to a common site; (b) inhibition by nucleotides occurs through simple reversible binding, as a consequence of tighter binding to the closed-channel relative to the open-channel conformation; (c) the conformation of the nucleotide binding site is not directly modulated by Ca2+ and voltage; (d) the differences in inhibitory potency of ATP, ADP, and AMP reflect their different affinities for the closed channel; and (e) though decavanadate is the only example found to date of a compound that stimulates Po with high affinity even in the presence of millimolar nucleotides, apparently by competing for the nucleotide binding site, a comparable mechanism might allow CA-NSC channels to open in living cells despite physiological levels of nucleotides. Decavanadate now provides a valuable tool for studying native CA-NSC channels and for screening cloned

  7. Transcriptional and Posttranslational Regulation of Nucleotide Excision Repair: The Guardian of the Genome against Ultraviolet Radiation

    PubMed Central

    Park, Jeong-Min; Kang, Tae-Hong

    2016-01-01

    Ultraviolet (UV) radiation from sunlight represents a constant threat to genome stability by generating modified DNA bases such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). If unrepaired, these lesions can have deleterious effects, including skin cancer. Mammalian cells are able to neutralize UV-induced photolesions through nucleotide excision repair (NER). The NER pathway has multiple components including seven xeroderma pigmentosum (XP) proteins (XPA to XPG) and numerous auxiliary factors, including ataxia telangiectasia and Rad3-related (ATR) protein kinase and RCC1 like domain (RLD) and homologous to the E6-AP carboxyl terminus (HECT) domain containing E3 ubiquitin protein ligase 2 (HERC2). In this review we highlight recent data on the transcriptional and posttranslational regulation of NER activity. PMID:27827925

  8. The Regulated Secretory Pathway and Human Disease: Insights from Gene Variants and Single Nucleotide Polymorphisms

    PubMed Central

    Lin, Wei-Jye; Salton, Stephen R.

    2013-01-01

    The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired. PMID:23964269

  9. The regulated secretory pathway and human disease: insights from gene variants and single nucleotide polymorphisms.

    PubMed

    Lin, Wei-Jye; Salton, Stephen R

    2013-01-01

    The regulated secretory pathway provides critical control of peptide, growth factor, and hormone release from neuroendocrine and endocrine cells, and neurons, maintaining physiological homeostasis. Propeptides and prohormones are packaged into dense core granules (DCGs), where they frequently undergo tissue-specific processing as the DCG matures. Proteins of the granin family are DCG components, and although their function is not fully understood, data suggest they are involved in DCG formation and regulated protein/peptide secretion, in addition to their role as precursors of bioactive peptides. Association of gene variation, including single nucleotide polymorphisms (SNPs), with neuropsychiatric, endocrine, and metabolic diseases, has implicated specific secreted proteins and peptides in disease pathogenesis. For example, a SNP at position 196 (G/A) of the human brain-derived neurotrophic factor gene dysregulates protein processing and secretion and leads to cognitive impairment. This suggests more generally that variants identified in genes encoding secreted growth factors, peptides, hormones, and proteins involved in DCG biogenesis, protein processing, and the secretory apparatus, could provide insight into the process of regulated secretion as well as disorders that result when it is impaired.

  10. The coiled-coil domain of zebrafish TRPM7 regulates Mg·nucleotide sensitivity

    PubMed Central

    Jansen, Chad; Sahni, Jaya; Suzuki, Sayuri; Horgen, F. David; Penner, Reinhold; Fleig, Andrea

    2016-01-01

    TRPM7 is a member of the Transient-Receptor-Potential Melastatin ion channel family. TRPM7 is a unique fusion protein of an ion channel and an α-kinase. Although mammalian TRPM7 is well characterized biophysically and its pivotal role in cancer, ischemia and cardiovascular disease is becoming increasingly evident, the study of TRPM7 in mouse models has been hampered by embryonic lethality of transgenic ablations. In zebrafish, functional loss of TRPM7 (drTRPM7) manifests itself in an array of non-lethal physiological malfunctions. Here, we investigate the regulation of wild type drTRPM7 and multiple C-terminal truncation mutants. We find that the biophysical properties of drTRPM7 are very similar to mammalian TRPM7. However, pharmacological profiling reveals that drTRPM7 is facilitated rather than inhibited by 2-APB, and that the TRPM7 inhibitor waixenicin A has no effect. This is reminiscent of the pharmacological profile of human TRPM6, the sister channel kinase of TRPM7. Furthermore, using truncation mutations, we show that the coiled-coil domain of drTRPM7 is involved in the channel’s regulation by magnesium (Mg) and Mg·adenosine triphosphate (Mg·ATP). We propose that drTRPM7 has two protein domains that regulate inhibition by intracellular magnesium and nucleotides, and one domain that is concerned with sensing magnesium only. PMID:27628598

  11. Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code.

    PubMed

    Hoernes, Thomas Philipp; Clementi, Nina; Faserl, Klaus; Glasner, Heidelinde; Breuker, Kathrin; Lindner, Herbert; Hüttenhofer, Alexander; Erlacher, Matthias David

    2016-01-29

    Nucleotide modifications within RNA transcripts are found in every organism in all three domains of life. 6-methyladeonsine (m(6)A), 5-methylcytosine (m(5)C) and pseudouridine (Ψ) are highly abundant nucleotide modifications in coding sequences of eukaryal mRNAs, while m(5)C and m(6)A modifications have also been discovered in archaeal and bacterial mRNAs. Employing in vitro translation assays, we systematically investigated the influence of nucleotide modifications on translation. We introduced m(5)C, m(6)A, Ψ or 2'-O-methylated nucleotides at each of the three positions within a codon of the bacterial ErmCL mRNA and analyzed their influence on translation. Depending on the respective nucleotide modification, as well as its position within a codon, protein synthesis remained either unaffected or was prematurely terminated at the modification site, resulting in reduced amounts of the full-length peptide. In the latter case, toeprint analysis of ribosomal complexes was consistent with stalling of translation at the modified codon. When multiple nucleotide modifications were introduced within one codon, an additive inhibitory effect on translation was observed. We also identified the m(5)C modification to alter the amino acid identity of the corresponding codon, when positioned at the second codon position. Our results suggest a novel mode of gene regulation by nucleotide modifications in bacterial mRNAs. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Genome-wide identification and analysis of rice genes preferentially expressed in pollen at an early developmental stage.

    PubMed

    Nguyen, Tien Dung; Moon, Sunok; Nguyen, Van Ngoc Tuyet; Gho, Yunsil; Chandran, Anil Kumar Nalini; Soh, Moon-Soo; Song, Jong Tae; An, Gynheung; Oh, Sung Aeong; Park, Soon Ki; Jung, Ki-Hong

    2016-09-01

    Microspore production using endogenous developmental programs has not been well studied. The main limitation is the difficulty in identifying genes preferentially expressed in pollen grains at early stages. To overcome this limitation, we collected transcriptome data from anthers and microspore/pollen and performed meta-expression analysis. Subsequently, we identified 410 genes showing preferential expression patterns in early developing pollen samples of both japonica and indica cultivars. The expression patterns of these genes are distinguishable from genes showing pollen mother cell or tapetum-preferred expression patterns. Gene Ontology enrichment and MapMan analyses indicated that microspores in rice are closely linked with protein degradation, nucleotide metabolism, and DNA biosynthesis and regulation, while the pollen mother cell or tapetum are strongly associated with cell wall metabolism, lipid metabolism, secondary metabolism, and RNA biosynthesis and regulation. We also generated transgenic lines under the control of the promoters of eight microspore-preferred genes and confirmed the preferred expression patterns in plants using the GUS reporting system. Furthermore, cis-regulatory element analysis revealed that pollen specific elements such as POLLEN1LELAT52, and 5659BOXLELAT5659 were commonly identified in the promoter regions of eight rice genes with more frequency than estimation. Our study will provide new sights on early pollen development in rice, a model crop plant.

  13. The regulator of calcineurin 1 increases adenine nucleotide translocator 1 and leads to mitochondrial dysfunctions.

    PubMed

    Jiang, Hui; Zhang, Chen; Tang, Yu; Zhao, Juan; Wang, Tan; Liu, Heng; Sun, Xiulian

    2017-01-01

    The over-expression of regulator of calcineurin 1 isoform 1 (RCAN1.1) has been implicated in mitochondrial dysfunctions of Alzheimer's disease; however, the mechanism linking RCAN1.1 over-expression and the mitochondrial dysfunctions remains unknown. In this study, we use human neuroblastoma SH-SY5Y cells stably expressing RCAN1.1S and rat primary neurons infected with RCAN1.1S expression lentivirus to study the association of RCAN1 with mitochondrial functions. Our study here showed that the over-expression of RCAN1.1S remarkably up-regulates the expression of adenine nucleotide translocator (ANT1) by stabilizing ANT1 mRNA. The increased ANT1 level leads to accelerated ATP-ADP exchange rate, more Ca(2+) -induced mitochondrial permeability transition pore opening, increased cytochrome c release, and eventually cell apoptosis. Furthermore, knockdown of ANT1 expression brings these mitochondria perturbations caused by RCAN1.1S back to normal. The effect of RCAN1.1S on ANT1 was independent of its inhibition on calcineurin. This study elucidated a novel function of RCAN1 in mitochondria and provides a molecular basis for the RCAN1.1S over-expression-induced mitochondrial dysfunctions and neuronal apoptosis. © 2016 The Authors. Journal of Neurochemistry published by John Wiley & Sons Ltd on behalf of International Society for Neurochemistry.

  14. Regulation and disregulation of mammalian nucleotide excision repair: A pathway to nongermline breast carcinogenesis

    DOE PAGES

    Latimer, Jean J.; Majekwana, Vongai J.; Pabon-Padin, Yashira R.; ...

    2014-12-19

    Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NERmore » pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this article, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.« less

  15. Regulation and disregulation of mammalian nucleotide excision repair: A pathway to nongermline breast carcinogenesis

    SciTech Connect

    Latimer, Jean J.; Majekwana, Vongai J.; Pabon-Padin, Yashira R.; Pimpley, Manasi R.; Grant, Stephen G.

    2014-12-19

    Nucleotide excision repair (NER) is important as a modulator of disease, especially in constitutive deficiencies, such as the cancer predisposition syndrome Xeroderma pigmentosum. We have found profound variation of NER capacity among normal individuals, between cell-types and during carcinogenesis. NER is a repair system for many types of DNA damage, and therefore many types of genotoxic carcinogenic exposures, including ultraviolet light, products of organic combustion, metals, oxidative stress, etc. Since NER is intimately related to cellular metabolism, requiring components of both the DNA replicative and transcription machinery, it has a narrow range of functional viability. Thus, genes in the NER pathway are expressed at the low levels manifested by, for example, nuclear transcription factors. Since NER activity and gene expression vary by cell-type, it is inherently epigenetically regulated. Furthermore, this epigenetic regulation is disregulated during sporadic breast carcinogenesis. Loss of NER is one basis of genomic instability, a required element in cellular transformation, and one that potentially modulates response to therapy. In this article, we demonstrate differences in NER capacity in eight adult mouse tissues, and place this result into the context of our previous work on mouse extraembryonic tissues, normal human tissues and sporadic early stage human breast cancer.

  16. Diverse roles of guanine nucleotide exchange factors in regulating collective cell migration.

    PubMed

    Zaritsky, Assaf; Tseng, Yun-Yu; Rabadán, M Angeles; Krishna, Shefali; Overholtzer, Michael; Danuser, Gaudenz; Hall, Alan

    2017-06-05

    Efficient collective migration depends on a balance between contractility and cytoskeletal rearrangements, adhesion, and mechanical cell-cell communication, all controlled by GTPases of the RHO family. By comprehensive screening of guanine nucleotide exchange factors (GEFs) in human bronchial epithelial cell monolayers, we identified GEFs that are required for collective migration at large, such as SOS1 and β-PIX, and RHOA GEFs that are implicated in intercellular communication. Down-regulation of the latter GEFs differentially enhanced front-to-back propagation of guidance cues through the monolayer and was mirrored by down-regulation of RHOA expression and myosin II activity. Phenotype-based clustering of knockdown behaviors identified RHOA-ARHGEF18 and ARHGEF3-ARHGEF28-ARHGEF11 clusters, indicating that the latter may signal through other RHO-family GTPases. Indeed, knockdown of RHOC produced an intermediate between the two phenotypes. We conclude that for effective collective migration, the RHOA-GEFs → RHOA/C → actomyosin pathways must be optimally tuned to compromise between generation of motility forces and restriction of intercellular communication. © 2017 Zaritsky et al.

  17. Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis.

    PubMed

    Nepal, Chirag; Hadzhiev, Yavor; Previti, Christopher; Haberle, Vanja; Li, Nan; Takahashi, Hazuki; Suzuki, Ana Maria M; Sheng, Ying; Abdelhamid, Rehab F; Anand, Santosh; Gehrig, Jochen; Akalin, Altuna; Kockx, Christel E M; van der Sloot, Antoine A J; van Ijcken, Wilfred F J; Armant, Olivier; Rastegar, Sepand; Watson, Craig; Strähle, Uwe; Stupka, Elia; Carninci, Piero; Lenhard, Boris; Müller, Ferenc

    2013-11-01

    Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.

  18. Guanine nucleotide exchange factor RABGEF1 regulates keratinocyte-intrinsic signaling to maintain skin homeostasis

    PubMed Central

    Marichal, Thomas; El Abbas, Sophie; Sibilano, Riccardo; Zurek, Oliwia; Reber, Laurent L.; Pirottin, Dimitri; Kim, Jinah; Chambon, Pierre; Roers, Axel; Antoine, Nadine; Kawakami, Yuko; Bureau, Fabrice; Tam, See-Ying; Tsai, Mindy

    2016-01-01

    Epidermal keratinocytes form a structural and immune barrier that is essential for skin homeostasis. However, the mechanisms that regulate epidermal barrier function are incompletely understood. Here we have found that keratinocyte-specific deletion of the gene encoding RAB guanine nucleotide exchange factor 1 (RABGEF1, also known as RABEX-5) severely impairs epidermal barrier function in mice and induces an allergic cutaneous and systemic phenotype. RABGEF1-deficient keratinocytes exhibited aberrant activation of the intrinsic IL-1R/MYD88/NF-κB signaling pathway and MYD88-dependent abnormalities in expression of structural proteins that contribute to skin barrier function. Moreover, ablation of MYD88 signaling in RABGEF1-deficient keratinocytes or deletion of Il1r1 restored skin homeostasis and prevented development of skin inflammation. We further demonstrated that epidermal RABGEF1 expression is reduced in skin lesions of humans diagnosed with either atopic dermatitis or allergic contact dermatitis as well as in an inducible mouse model of allergic dermatitis. Our findings reveal a key role for RABGEF1 in dampening keratinocyte-intrinsic MYD88 signaling and sustaining epidermal barrier function in mice, and suggest that dysregulation of RABGEF1 expression may contribute to epidermal barrier dysfunction in allergic skin disorders in mice and humans. Thus, RABGEF1-mediated regulation of IL-1R/MYD88 signaling might represent a potential therapeutic target. PMID:27820702

  19. Human Aldo-Keto Reductases: Function, Gene Regulation, and Single Nucleotide Polymorphisms

    PubMed Central

    Penning, Trevor M.; Drury, Jason E.

    2007-01-01

    Aldo-Keto Reductases (AKRs) are a superfamily of NAD(P)H linked oxidoreductases that are generally monomeric 34- 37 kDa proteins present in all phyla. The superfamily consists of 15 families, which contains 151 members (www.med.upenn.edu/akr). Thirteen human AKRs exist that use endogenous substrates (sugar and lipid aldehydes, prostaglandins, retinals and steroid hormones), and in many instances they regulate nuclear receptor signaling. Exogenous substrates include metabolites implicated in chemical carcinogenesis: NNK (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), polycyclic aromatic hydrocarbon trans-dihydrodiols, and aflatoxin dialdehyde. Promoter analysis of the human genes identifies common elements involved in their regulation which include osmotic response elements, antioxidant response elements, xenobiotic response elements, AP-1 sites and steroid response elements. The human AKRs are highly polymorphic, and in some instances single nucleotide polymorphisms (SNPs) of high penetrance exist. This suggests that there will be inter-individual variation in endogenous and xenobiotic metabolism which in turn affect susceptibility to nuclear receptor signaling and chemical carcinogenesis. PMID:17537398

  20. Dynamic regulation of the transcription initiation landscape at single nucleotide resolution during vertebrate embryogenesis

    PubMed Central

    Nepal, Chirag; Hadzhiev, Yavor; Previti, Christopher; Haberle, Vanja; Li, Nan; Takahashi, Hazuki; Suzuki, Ana Maria M.; Sheng, Ying; Abdelhamid, Rehab F.; Anand, Santosh; Gehrig, Jochen; Akalin, Altuna; Kockx, Christel E.M.; van der Sloot, Antoine A.J.; van IJcken, Wilfred F.J.; Armant, Olivier; Rastegar, Sepand; Watson, Craig; Strähle, Uwe; Stupka, Elia; Carninci, Piero; Lenhard, Boris; Müller, Ferenc

    2013-01-01

    Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates. PMID:24002785

  1. An Actin-Binding Protein, LlLIM1, Mediates Calcium and Hydrogen Regulation of Actin Dynamics in Pollen Tubes1[C][W][OA

    PubMed Central

    Wang, Huei-Jing; Wan, Ai-Ru; Jauh, Guang-Yuh

    2008-01-01

    Actin microfilaments are crucial for polar cell tip growth, and their configurations and dynamics are regulated by the actions of various actin-binding proteins (ABPs). We explored the function of a lily (Lilium longiflorum) pollen-enriched LIM domain-containing protein, LlLIM1, in regulating the actin dynamics in elongating pollen tube. Cytological and biochemical assays verified LlLIM1 functioning as an ABP, promoting filamentous actin (F-actin) bundle assembly and protecting F-actin against latrunculin B-mediated depolymerization. Overexpressed LlLIM1 significantly disturbed pollen tube growth and morphology, with multiple tubes protruding from one pollen grain and coaggregation of FM4-64-labeled vesicles and Golgi apparatuses at the subapex of the tube tip. Moderate expression of LlLIM1 induced an oscillatory formation of asterisk-shaped F-actin aggregates that oscillated with growth period but in different phases at the subapical region. These results suggest that the formation of LlLIM1-mediated overstabilized F-actin bundles interfered with endomembrane trafficking to result in growth retardation. Cosedimentation assays revealed that the binding affinity of LlLIM1 to F-actin was simultaneously regulated by both pH and Ca2+: LlLIM1 showed a preference for F-actin binding under low pH and low Ca2+ concentration. The potential functions of LlLIM1 as an ABP sensitive to pH and calcium in integrating endomembrane trafficking, oscillatory pH, and calcium circumstances to regulate tip-focused pollen tube growth are discussed. PMID:18480376

  2. Mixed Mating System Are Regulated by Fecundity in Shorea curtisii (Dipterocarpaceae) as Revealed by Comparison under Different Pollen Limited Conditions

    PubMed Central

    Tani, Naoki; Tsumura, Yoshihiko; Fukasawa, Keita; Kado, Tomoyuki; Taguchi, Yuriko; Lee, Soon Leong; Lee, Chai Ting; Muhammad, Norwati; Niiyama, Kaoru; Otani, Tatsuya; Yagihashi, Tsutomu; Tanouchi, Hiroyuki; Ripin, Azizi; Kassim, Abdul Rahman

    2015-01-01

    The maintenance of mixed mating was studied in Shorea curtisii, a dominant and widely distributed dipterocarp species in Southeast Asia. Paternity and hierarchical Bayesian analyses were used to estimate the parameters of pollen dispersal kernel, male fecundity and self-pollen affinity. We hypothesized that partial self incompatibility and/or inbreeding depression reduce the number of selfed seeds if the mother trees receive sufficient pollen, whereas reproductive assurance increases the numbers of selfed seeds under low amounts of pollen. Comparison of estimated parameters of self-pollen affinity between high density undisturbed and low density selectively logged forests indicated that self-pollen was selectively excluded from mating in the former, probably due to partial self incompatibility or inbreeding depression until seed maturation. By estimating the self-pollen affinity of each mother tree in both forests, mother trees with higher amount of self-pollen indicated significance of self-pollen affinity with negative estimated value. The exclusion of self-fertilization and/or inbreeding depression during seed maturation occurred in the mother trees with large female fecundity, whereas reproductive assurance increased self-fertilization in the mother trees with lower female fecundity. PMID:25938512

  3. The atypical Guanine-nucleotide exchange factor, dock7, negatively regulates schwann cell differentiation and myelination.

    PubMed

    Yamauchi, Junji; Miyamoto, Yuki; Hamasaki, Hajime; Sanbe, Atsushi; Kusakawa, Shinji; Nakamura, Akane; Tsumura, Hideki; Maeda, Masahiro; Nemoto, Noriko; Kawahara, Katsumasa; Torii, Tomohiro; Tanoue, Akito

    2011-08-31

    In development of the peripheral nervous system, Schwann cells proliferate, migrate, and ultimately differentiate to form myelin sheath. In all of the myelination stages, Schwann cells continuously undergo morphological changes; however, little is known about their underlying molecular mechanisms. We previously cloned the dock7 gene encoding the atypical Rho family guanine-nucleotide exchange factor (GEF) and reported the positive role of Dock7, the target Rho GTPases Rac/Cdc42, and the downstream c-Jun N-terminal kinase in Schwann cell migration (Yamauchi et al., 2008). We investigated the role of Dock7 in Schwann cell differentiation and myelination. Knockdown of Dock7 by the specific small interfering (si)RNA in primary Schwann cells promotes dibutyryl cAMP-induced morphological differentiation, indicating the negative role of Dock7 in Schwann cell differentiation. It also results in a shorter duration of activation of Rac/Cdc42 and JNK, which is the negative regulator of myelination, and the earlier activation of Rho and Rho-kinase, which is the positive regulator of myelination. To obtain the in vivo evidence, we generated Dock7 short hairpin (sh)RNA transgenic mice. They exhibited a decreased expression of Dock7 in the sciatic nerves and enhanced myelin thickness, consistent with in vitro observation. The effects of the in vivo knockdown on the signals to Rho GTPases are similar to those of the in vitro knockdown. Collectively, the signaling through Dock7 negatively regulates Schwann cell differentiation and the onset of myelination, demonstrating the unexpected role of Dock7 in the interplay between Schwann cell migration and myelination.

  4. Numb regulates the polarized delivery of cyclic nucleotide-gated ion channels in rod photoreceptor cilia.

    PubMed

    Ramamurthy, Vasanth; Jolicoeur, Christine; Koutroumbas, Demetra; Mühlhans, Johanna; Le, Yun-Zheng; Hauswirth, William W; Giessl, Andreas; Cayouette, Michel

    2014-10-15

    The development and maintenance of protein compartmentalization is essential for neuronal function. A striking example is observed in light-sensing photoreceptors, in which the apical sensory cilium is subdivided into an inner and outer segment, each containing specific proteins essential for vision. It remains unclear, however, how such polarized protein localization is regulated. We report here that the endocytic adaptor protein Numb localizes to the inner, but not the outer segment of mouse photoreceptor cilia. Rod photoreceptor-specific inactivation of numb in vivo leads to progressive photoreceptor degeneration, indicating an essential role for Numb in photoreceptor cell biology. Interestingly, we report that loss of Numb in photoreceptors does not affect the localization of outer segment disk membrane proteins, such as rhodopsin, Peripherin-rds, Rom-1, and Abca4, but significantly disrupts the localization of the rod cyclic nucleotide-gated (Cng) channels, which accumulates on the inner segment plasma membrane in addition to its normal localization to the outer segments. Mechanistically, we show that Numb interacts with both subunits of the Cng channel and promotes the trafficking of Cnga1 to the recycling endosome. These results suggest a model in which Numb prevents targeting of Cng channels to the inner segment, by promoting their trafficking through the recycling endosome, where they can be sorted for specific delivery to the outer segment. This study uncovers a novel mechanism regulating polarized protein delivery in light-sensing cilia, raising the possibility that Numb plays a part in the regulation of protein trafficking in other types of cilia. Copyright © 2014 the authors 0270-6474/14/3413976-12$15.00/0.

  5. Roles of phosphodiesterases in the regulation of the cardiac cyclic nucleotide cross-talk signaling network.

    PubMed

    Zhao, Claire Y; Greenstein, Joseph L; Winslow, Raimond L

    2016-02-01

    The balanced signaling between the two cyclic nucleotides (cNs) cAMP and cGMP plays a critical role in regulating cardiac contractility. Their degradation is controlled by distinctly regulated phosphodiesterase isoenzymes (PDEs), which in turn are also regulated by these cNs. As a result, PDEs facilitate communication between the β-adrenergic and Nitric Oxide (NO)/cGMP/Protein Kinase G (PKG) signaling pathways, which regulate the synthesis of cAMP and cGMP respectively. The phenomena in which the cAMP and cGMP pathways influence the dynamics of each other are collectively referred to as cN cross-talk. However, the cross-talk response and the individual roles of each PDE isoenzyme in shaping this response remain to be fully characterized. We have developed a computational model of the cN cross-talk network that mechanistically integrates the β-adrenergic and NO/cGMP/PKG pathways via regulation of PDEs by both cNs. The individual model components and the integrated network model replicate experimentally observed activation-response relationships and temporal dynamics. The model predicts that, due to compensatory interactions between PDEs, NO stimulation in the presence of sub-maximal β-adrenergic stimulation results in an increase in cytosolic cAMP accumulation and corresponding increases in PKA-I and PKA-II activation; however, the potentiation is small in magnitude compared to that of NO activation of the NO/cGMP/PKG pathway. In a reciprocal manner, β-adrenergic stimulation in the presence of sub-maximal NO stimulation results in modest cGMP elevation and corresponding increase in PKG activation. In addition, we demonstrate that PDE2 hydrolyzes increasing amounts of cAMP with increasing levels of β-adrenergic stimulation, and hydrolyzes increasing amounts of cGMP with decreasing levels of NO stimulation. Finally, we show that PDE2 compensates for inhibition of PDE5 both in terms of cGMP and cAMP dynamics, leading to cGMP elevation and increased PKG activation

  6. PLC-Mediated Signaling Pathway in Pollen Tubes Regulates the Gametophytic Self-incompatibility of Pyrus Species.

    PubMed

    Qu, Haiyong; Guan, Yaqin; Wang, Yongzhang; Zhang, Shaolin

    2017-01-01

    Among the Rosaceae species, the gametophytic self-incompatibility (GSI) is controlled by a single multi-allelic S locus, which is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), which is essential for identifying self-pollen. However, the S-RNase system has not been fully characterized. In this study, the self-S-RNase inhibited the Ca(2+)-permeable channel activity at pollen tube apices and the selectively decreased phospholipase C (PLC) activity in the plasma membrane of Pyrus pyrifolia pollen tubes. Self-S-RNase decreased the Ca(2+) influx through a PLC-mediated signaling pathway. Phosphatidylinositol-specific PLC has a 26-amino acid insertion in pollen tubes of the 'Jinzhuili' cultivar, which is a spontaneous self-compatible mutant of the 'Yali' cultivar. 'Yali' plants exhibit a typical S-RNase-based GSI. Upon self-pollination, PLC gene expression is significantly higher in 'Jinzhuili' pollen tubes than that in 'Yali' pollen tubes. Moreover, the PLC in pollen tubes can only interact with one of the two types of S-RNase from the style. In the Pyrus x bretschneideri Rehd, the PLC directly interacted with the S7-RNase in the pollen tube, but not with the S34-RNase. Collectively, our results reveal that the effects of S-RNase on PLC activity are required for S-specific pollen rejection, and that PLC-IP3 participates in the self-incompatibility reaction of Pyrus species.

  7. PLC-Mediated Signaling Pathway in Pollen Tubes Regulates the Gametophytic Self-incompatibility of Pyrus Species

    PubMed Central

    Qu, Haiyong; Guan, Yaqin; Wang, Yongzhang; Zhang, Shaolin

    2017-01-01

    Among the Rosaceae species, the gametophytic self-incompatibility (GSI) is controlled by a single multi-allelic S locus, which is composed of the pistil-S and pollen-S genes. The pistil-S gene encodes a polymorphic ribonuclease (S-RNase), which is essential for identifying self-pollen. However, the S-RNase system has not been fully characterized. In this study, the self-S-RNase inhibited the Ca2+-permeable channel activity at pollen tube apices and the selectively decreased phospholipase C (PLC) activity in the plasma membrane of Pyrus pyrifolia pollen tubes. Self-S-RNase decreased the Ca2+ influx through a PLC-mediated signaling pathway. Phosphatidylinositol-specific PLC has a 26-amino acid insertion in pollen tubes of the ‘Jinzhuili’ cultivar, which is a spontaneous self-compatible mutant of the ‘Yali’ cultivar. ‘Yali’ plants exhibit a typical S-RNase-based GSI. Upon self-pollination, PLC gene expression is significantly higher in ‘Jinzhuili’ pollen tubes than that in ‘Yali’ pollen tubes. Moreover, the PLC in pollen tubes can only interact with one of the two types of S-RNase from the style. In the Pyrus x bretschneideri Rehd, the PLC directly interacted with the S7-RNase in the pollen tube, but not with the S34-RNase. Collectively, our results reveal that the effects of S-RNase on PLC activity are required for S-specific pollen rejection, and that PLC-IP3 participates in the self-incompatibility reaction of Pyrus species. PMID:28729872

  8. Basic helix-loop-helix transcription factor BcbHLHpol functions as a positive regulator of pollen development in non-heading Chinese cabbage.

    PubMed

    Liu, Tongkun; Li, Ying; Zhang, Changwei; Duan, Weike; Huang, Feiyi; Hou, Xilin

    2014-12-01

    Cytoplasmic male sterility (CMS) is a common trait in higher plants, and several transcription factors regulate pollen development. Previously, we obtained a basic helix-loop-helix transcription factor, BcbHLHpol, via suppression subtractive hybridization in non-heading Chinese cabbage. However, the regulatory function of BcbHLHpol during anther and pollen development remains unclear. In this study, BcbHLHpol was cloned, and its tissue-specific expression profile was analyzed. The results of real-time polymerase chain reaction showed that BcbHLHpol was highly expressed in maintainer buds and that the transcripts of BcbHLHpol significantly decreased in the buds of pol CMS. A virus-induced gene silencing vector that targets BcbHLHpol was constructed and transformed into Brassica campestris plants to further explore the function of BcbHLHpol. Male sterility and short stature were observed in BcbHLHpol-silenced plants. The degradation of tapetal cells was inhibited in BcbHLHpol-silenced plants, and nutrients were insufficiently supplied to the microspore. These phenomena resulted in pollen abortion. This result indicates that BcbHLHpol functions as a positive regulator in pollen development. Yeast two-hybrid and bimolecular fluorescence complementation assays revealed that BcbHLHpol interacted with BcSKP1 in the nucleus. This finding suggests that BcbHLHpol and BcSKP1 are positive coordinating regulators of pollen development. Quantitative real-time PCR indicated that BcbHLHpol and BcSKP1 can be induced at low temperatures. Thus, we propose that BcbHLHpol is necessary for meiosis. This study provides insights into the regulatory functions of the BcbHLHpol network during anther development.

  9. SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair

    PubMed Central

    Akita, Masaki; Tak, Yon-Soo; Shimura, Tsutomu; Matsumoto, Syota; Okuda-Shimizu, Yuki; Shimizu, Yuichiro; Nishi, Ryotaro; Saitoh, Hisato; Iwai, Shigenori; Mori, Toshio; Ikura, Tsuyoshi; Sakai, Wataru; Hanaoka, Fumio; Sugasawa, Kaoru

    2015-01-01

    The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER. PMID:26042670

  10. Cyclic nucleotide gated channel 10 negatively regulates salt tolerance by mediating Na+ transport in Arabidopsis.

    PubMed

    Jin, Yakang; Jing, Wen; Zhang, Qun; Zhang, Wenhua

    2015-01-01

    A number of cyclic nucleotide gated channel (CNGC) genes have been identified in plant genomes, but their functions are mainly undefined. In this study, we identified the role of CNGC10 in the response of Arabidopsis thaliana to salt stress. The cngc10 T-DNA insertion mutant showed greater tolerance to salt than wild-type A. thaliana during seed germination and seedling growth. The cngc10 mutant accumulated less Na(+) and K(+), but not less Ca(2+), in shoots in response to salt stress. By contrast, overexpression of CNGC10 resulted in greater sensitivity to salt stress, and complementation of this gene recovered salt sensitivity. In response to salt stress, heterologous expression of CNGC10 in the Na(+) sensitive yeast mutant strain B31 inhibited growth due to accumulation of Na(+) at a rate greater than that of yeast transformed with an empty vector. Quantitative RT-PCR analysis demonstrated that CNGC10 was expressed mainly in roots and flowers. GUS analysis of a root cross section indicated that CNGC10 was expressed mainly in the endodermis and epidermis. Furthermore, the expression of CNGC10 in roots was dramatically inhibited by exposure to 200 mM NaCl for 6 h. These data suggest that CNGC10 negatively regulates salt tolerance in A. thaliana and may be involved in mediating Na(+) transport.

  11. The Cyclic Nucleotide-Gated Channel CNGC14 Regulates Root Gravitropism in Arabidopsis thaliana.

    PubMed

    Shih, Han-Wei; DePew, Cody L; Miller, Nathan D; Monshausen, Gabriele B

    2015-12-07

    In plant roots, auxin inhibits cell expansion, and an increase in cellular auxin levels on the lower flanks of gravistimulated roots suppresses growth and thereby causes downward bending. These fundamental features of root growth responses to auxin were first described over 80 years ago, but our understanding of the underlying molecular mechanisms has remained scant. Here, we report that CYCLIC NUCLEOTIDE-GATED CHANNEL 14 (CNGC14) is essential for the earliest phase of auxin-induced ion signaling and growth inhibition in Arabidopsis roots. Using a fluorescence-imaging-based genetic screen, we found that cngc14 mutants exhibit a complete loss of rapid Ca(2+) and pH signaling in response to auxin treatment. Similarly impaired ion signaling was observed upon gravistimulation. We further developed a kinematic analysis approach to study dynamic root growth responses to auxin at high spatiotemporal resolution. These analyses revealed that auxin-induced growth inhibition and gravitropic bending are significantly delayed in cngc14 compared to wild-type roots, where auxin suppresses cell expansion within 1 min of treatment. Finally, we demonstrate that auxin-induced cytosolic Ca(2+) changes are required for rapid growth inhibition. Our results support a direct role for CNGC14-dependent Ca(2+) signaling in regulating the early posttranscriptional phase of auxin growth responses in Arabidopsis roots. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

    PubMed Central

    Leonard, John D.; Narlikar, Geeta J.

    2015-01-01

    SUMMARY The ATP-dependent chromatin assembly factor (ACF) is a dimeric motor that spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes. PMID:25684208

  13. Guanyl Nucleotide Exchange Factor Sql2 and Ras2 Regulate Filamentous Growth in Ustilago maydis

    PubMed Central

    Müller, Philip; Katzenberger, Jörg D.; Loubradou , Gabriel; Kahmann, Regine

    2003-01-01

    The cyclic AMP (cAMP)-signaling pathway regulates cell morphology and plays a crucial role during pathogenic development of the plant-pathogenic fungus Ustilago maydis. Strains lacking components of this signaling pathway, such as the Gα-subunit Gpa3 or the adenylyl cyclase Uac1, are nonpathogenic and grow filamentously. On the other hand, strains exhibiting an activated cAMP pathway due to a dominant-active allele of gpa3 display a glossy colony phenotype and are unable to proliferate in plant tumors. Here we present the identification of sql2 as a suppressor of the glossy colony phenotype of a gpa3Q206L strain. sql2 encodes a protein with similarity to CDC25-like guanine nucleotide exchange factors, which are known to act on Ras proteins. Overexpression of sql2 leads to filamentous growth that cannot be suppressed by exogenous cAMP, suggesting that Sql2 does not act upstream of Uac1. To gain more insight in signaling processes regulated by Sql2, we isolated two genes encoding Ras proteins. Expression of dominant active alleles of ras1 and ras2 showed that Ras2 induces filamentous growth while Ras1 does not affect cell morphology but elevates pheromone gene expression. These results indicate that Ras1 and Ras2 fulfill different functions in U. maydis. Moreover, observed similarities between the filaments induced by sql2 and ras2 suggest that Sql2 is an activator of Ras2. Interestingly, sql2 deletion mutants are affected in pathogenic development but not in mating, indicating a specific function of sql2 during pathogenesis. PMID:12796306

  14. Alternative Splicing Governs Cone Cyclic Nucleotide-gated (CNG) Channel Sensitivity to Regulation by Phosphoinositides*

    PubMed Central

    Dai, Gucan; Sherpa, Tshering; Varnum, Michael D.

    2014-01-01

    Precursor mRNA encoding CNGA3 subunits of cone photoreceptor cyclic nucleotide-gated (CNG) channels undergoes alternative splicing, generating isoforms differing in the N-terminal cytoplasmic region of the protein. In humans, four variants arise from alternative splicing, but the functional significance of these changes has been a persistent mystery. Heterologous expression of the four possible CNGA3 isoforms alone or with CNGB3 subunits did not reveal significant differences in basic channel properties. However, inclusion of optional exon 3, with or without optional exon 5, produced heteromeric CNGA3 + CNGB3 channels exhibiting an ∼2-fold greater shift in K1/2,cGMP after phosphatidylinositol 4,5-biphosphate or phosphatidylinositol 3,4,5-trisphosphate application compared with channels lacking the sequence encoded by exon 3. We have previously identified two structural features within CNGA3 that support phosphoinositides (PIPn) regulation of cone CNG channels: N- and C-terminal regulatory modules. Specific mutations within these regions eliminated PIPn sensitivity of CNGA3 + CNGB3 channels. The exon 3 variant enhanced the component of PIPn regulation that depends on the C-terminal region rather than the nearby N-terminal region, consistent with an allosteric effect on PIPn sensitivity because of altered N-C coupling. Alternative splicing of CNGA3 occurs in multiple species, although the exact variants are not conserved across CNGA3 orthologs. Optional exon 3 appears to be unique to humans, even compared with other primates. In parallel, we found that a specific splice variant of canine CNGA3 removes a region of the protein that is necessary for high sensitivity to PIPn. CNGA3 alternative splicing may have evolved, in part, to tune the interactions between cone CNG channels and membrane-bound phosphoinositides. PMID:24675082

  15. Differentiation-dependent p53 regulation of nucleotide excision repair in keratinocytes.

    PubMed Central

    Li, G.; Ho, V. C.; Mitchell, D. L.; Trotter, M. J.; Tron, V. A.

    1997-01-01

    The role of the tumor suppressor p53 in repair of ultraviolet light (UV)-induced DNA damage was evaluated using a host-cell reactivation (HCR) assay. HCR determines a cell's ability to repair UV-damaged DNA through reactivation of a transfected CAT reported plasmid. Most UV damage is removed through nucleotide excision repair (NER). Primary murine keratinocytes isolated from p53-deficient and wild-type p53 mice were used in the HCR assay. The NER was reduced in p53-/- keratinocytes as compared with p53+/+ keratinocytes. The reduced DNA repair in p53-/- mice was confirmed with a radioimmunoassay comparing cyclobutane dimers (CPDs) and (6-4) photoproducts in p53+/+ and p53-/- keratinocytes after the cells were exposed to UV irradiation. Our results demonstrate that wildtype p53 plays a significant role in regulating NER. Furthermore, as there is evidence that p53 protein levels decrease after keratinocytes become differentiated, we sought to determine whether p53 plays a role in NER in differentiated keratinocytes. Differentiation of the keratinocytes by increasing the Ca2+ concentration in the culture media resulted in a marked reduction in NER equally in both p53+/+ and p53-/- groups. This finding suggests that reduced DNA repair after differentiation is p53 independent. A similar reduction in HCR was confirmed in differentiated human keratinocytes. These data, taken together, indicate that p53 or p53-regulated proteins enhance NER in basal undifferentiated keratinocytes but not in differentiated cells. As nonmelanoma skin cancers originate from the basal keratinocytes, our findings suggest that loss of p53 may contribute to the pathogenesis of this common skin cancer. PMID:9095000

  16. Phosphodiesterases: Regulators of cyclic nucleotide signals and novel molecular target for movement disorders.

    PubMed

    Sharma, Sorabh; Kumar, Kushal; Deshmukh, Rahul; Sharma, Pyare Lal

    2013-08-15

    Movement disorders rank among the most common neurological disorders. During the last two decades substantial progress has been made in understanding of the pathological basis of these disorders. Although, several mechanisms have been proposed, downregulation of cyclic nucleotide mediated signaling cascade has consistently been shown to contribute to the striatal dysfunctioning as seen in movement disorders. Thus, counteracting dysregulated cyclic nucleotide signaling has been considered to be beneficial in movement disorders. Cyclic nucleotide phosphodiesterases (PDEs) are the enzymes responsible for the breakdown of cyclic nucleotides and upregulation in PDE activity has been reported in various movement disorders. Thus, PDE inhibition is considered to be a novel strategy to restore cerebral cyclic nucleotide levels and their downstream signalling cascade. Indeed, various PDE inhibitors have been tested pre-clinically and were reported to be neuroprotective in various neurodegenerative disorders associated with movement disabilities. In this review, we have discussed a putative role of PDE inhibitors in movement disorders and associated abnormalities.

  17. Involvement of the ribose operon repressor RbsR in regulation of purine nucleotide synthesis in Escherichia coli.

    PubMed

    Shimada, Tomohiro; Kori, Ayako; Ishihama, Akira

    2013-07-01

    Escherichia coli is able to utilize d-ribose as its sole carbon source. The genes for the transport and initial-step metabolism of d-ribose form a single rbsDACBK operon. RbsABC forms the ABC-type high-affinity d-ribose transporter, while RbsD and RbsK are involved in the conversion of d-ribose into d-ribose 5-phosphate. In the absence of inducer d-ribose, the ribose operon is repressed by a LacI-type transcription factor RbsR, which is encoded by a gene located downstream of this ribose operon. At present, the rbs operon is believed to be the only target of regulation by RbsR. After Genomic SELEX screening, however, we have identified that RbsR binds not only to the rbs promoter but also to the promoters of a set of genes involved in purine nucleotide metabolism. Northern blotting analysis indicated that RbsR represses the purHD operon for de novo synthesis of purine nucleotide but activates the add and udk genes involved in the salvage pathway of purine nucleotide synthesis. Taken together, we propose that RbsR is a global regulator for switch control between the de novo synthesis of purine nucleotides and its salvage pathway.

  18. Epigenetic reprogramming and small RNA silencing of transposable elements in pollen

    PubMed Central

    Slotkin, R. Keith; Vaughn, Matthew; Tanurdžic, Miloš; Borges, Filipe; Becker, Jörg D.; Feijó, José A.; Martienssen, Robert A.

    2009-01-01

    Summary The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that would transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with down-regulation of the heterochromatin remodeler DECREASE IN DNA METHYLATION 1 and of most TE siRNAs. However, 21 nucleotide siRNA from Athila retrotransposons is generated in pollen and accumulates in sperm, indicating that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes. PMID:19203581

  19. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice.

    PubMed

    Li, Xiang; Shahid, Muhammad Qasim; Wu, Jinwen; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2016-04-12

    MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice.

  20. Comparative Small RNA Analysis of Pollen Development in Autotetraploid and Diploid Rice

    PubMed Central

    Li, Xiang; Shahid, Muhammad Qasim; Wu, Jinwen; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2016-01-01

    MicroRNAs (miRNAs) play key roles in plant reproduction. However, knowledge on microRNAome analysis in autotetraploid rice is rather limited. Here, high-throughput sequencing technology was employed to analyze miRNAomes during pollen development in diploid and polyploid rice. A total of 172 differentially expressed miRNAs (DEM) were detected in autotetraploid rice compared to its diploid counterpart, and 57 miRNAs were specifically expressed in autotetraploid rice. Of the 172 DEM, 115 and 61 miRNAs exhibited up- and down-regulation, respectively. Gene Ontology analysis on the targets of up-regulated DEM showed that they were enriched in transport and membrane in pre-meiotic interphase, reproduction in meiosis, and nucleotide binding in single microspore stage. osa-miR5788 and osa-miR1432-5p_R+1 were up-regulated in meiosis and their targets revealed interaction with the meiosis-related genes, suggesting that they may involve in the genes regulation associated with the chromosome behavior. Abundant 24 nt siRNAs associated with transposable elements were found in autotetraploid rice during pollen development; however, they significantly declined in diploid rice, suggesting that 24 nt siRNAs may play a role in pollen development. These findings provide a foundation for understanding the effect of polyploidy on small RNA expression patterns during pollen development that cause pollen sterility in autotetraploid rice. PMID:27077850

  1. Fountain streaming contributes to fast tip-growth through regulating the gradients of turgor pressure and concentration in pollen tubes.

    PubMed

    Liu, ShaoBao; Liu, Han; Feng, ShangSheng; Lin, Min; Xu, Feng; Lu, Tian Jian

    2017-03-29

    Fountain streaming is a typical microfluidic pattern in plant cells, especially for cells with a high aspect ratio such as pollen tubes. Although it has been found that fountain streaming plays crucial roles in the transport of nutrients and metabolites, the positioning of organelles and the mixing of cytoplasms, its implications for the fast tip growth of pollen tubes remain a mystery. To address this, based on the observations of asiatic lily Lilium Casablanca, we developed physical models for reverse fountain streaming in pollen tubes and solved the hydrodynamics and advection-diffusion dynamics of viscous Stokes flow in the shank and apical region of pollen tubes. Theoretical and numerical results demonstrated that the gradients of turgor pressure and concentration of wall materials along the length of pollen tubes provide undamped driving force and high-efficiency materials supply, which are supposed to contribute to the fast tip-growth of pollen tubes. The sample experimental results show that the tip-growth will be abnormal when the gradients of turgor pressure change under osmotic stress induced by different concentrations of PEG-6000 (a dehydrant).

  2. CFTR: the nucleotide binding folds regulate the accessibility and stability of the activated state

    PubMed Central

    1996-01-01

    The functional roles of the two nucleotide binding folds, NBF1 and NBF2, in the activation of the cystic fibrosis transmembrane conductance regulator (CFTR) were investigated by measuring the rates of activation and deactivation of CFTR Cl- conductance in Xenopus oocytes. Activation of wild-type CFTR in response to application of forskolin and 3-isobutyl-1-methylxanthine (IBMX) was described by a single exponential. Deactivation after washout of the cocktail consisted of two phases: an initial slow phase, described by a latency, and an exponential decline. Rate analysis of CFTR variants bearing analogous mutations in NBF1 and NBF2 permitted us to characterize amino acid substitutions according to their effects on the accessibility and stability of the active state. Access to the active state was very sensitive to substitutions for the invariant glycine (G551) in NBF1, where mutations to alanine (A), serine (S), or aspartic acid (D) reduced the apparent on rate by more than tenfold. The analogous substitutions in NBF2 (G1349) also reduced the on rate, by twofold to 10-fold, but substantially destabilized the active state as well, as judged by increased deactivation rates. In the putative ATP-binding pocket of either NBF, substitution of alanine, glutamine (Q), or arginine (R) for the invariant lysine (K464 or K1250) reduced the on rate similarly, by two- to fourfold. In contrast, these analogous substitutions produced opposite effects on the deactivation rate. NBF1 mutations destabilized the active state, whereas the analogous substitutions in NBF2 stabilized the active state such that activation was prolonged compared with that seen with wild-type CFTR. Substitution of asparagine (N) for a highly conserved aspartic acid (D572) in the ATP-binding pocket of NBF1 dramatically slowed the on rate and destabilized the active state. In contrast, the analogous substitution in NBF2 (D1370N) did not appreciably affect the on rate and markedly stabilized the active state

  3. MoD Tools: regulatory motif discovery in nucleotide sequences from co-regulated or homologous genes

    PubMed Central

    Pavesi, Giulio; Mereghetti, Paolo; Zambelli, Federico; Stefani, Marco; Mauri, Giancarlo; Pesole, Graziano

    2006-01-01

    Understanding the complex mechanisms regulating gene expression at the transcriptional and post-transcriptional levels is one of the greatest challenges of the post-genomic era. The MoD (MOtif Discovery) Tools web server comprises a set of tools for the discovery of novel conserved sequence and structure motifs in nucleotide sequences, motifs that in turn are good candidates for regulatory activity. The server includes the following programs: Weeder, for the discovery of conserved transcription factor binding sites (TFBSs) in nucleotide sequences from co-regulated genes; WeederH, for the discovery of conserved TFBSs and distal regulatory modules in sequences from homologous genes; RNAProfile, for the discovery of conserved secondary structure motifs in unaligned RNA sequences whose secondary structure is not known. In this way, a given gene can be compared with other co-regulated genes or with its homologs, or its mRNA can be analyzed for conserved motifs regulating its post-transcriptional fate. The web server thus provides researchers with different strategies and methods to investigate the regulation of gene expression, at both the transcriptional and post-transcriptional levels. Available at and . PMID:16845071

  4. Cellular Pathways Regulating Responses to Compatible and Self-Incompatible Pollen in Brassica and Arabidopsis Stigmas Intersect at Exo70A1, a Putative Component of the Exocyst Complex[W

    PubMed Central

    Samuel, Marcus A.; Chong, Yolanda T.; Haasen, Katrina E.; Aldea-Brydges, May Grace; Stone, Sophia L.; Goring, Daphne R.

    2009-01-01

    In the Brassicaceae, compatible pollen–pistil interactions result in pollen adhesion to the stigma, while pollen grains from unrelated plant species are largely ignored. There can also be an additional layer of recognition to prevent self-fertilization, the self-incompatibility response, whereby self pollen grains are distinguished from nonself pollen grains and rejected. This pathway is activated in the stigma and involves the ARM repeat–containing 1 (ARC1) protein, an E3 ubiquitin ligase. In a screen for ARC1-interacting proteins, we have identified Brassica napus Exo70A1, a putative component of the exocyst complex that is known to regulate polarized secretion. We show through transgenic studies that loss of Exo70A1 in Brassica and Arabidopsis thaliana stigmas leads to the rejection of compatible pollen at the same stage as the self-incompatibility response. A red fluorescent protein:Exo70A1 fusion rescues this stigmatic defect in Arabidopsis and is found to be mobilized to the plasma membrane concomitant with flowers opening. By contrast, increased expression of Exo70A1 in self-incompatible Brassica partially overcomes the self pollen rejection response. Thus, our data show that the Exo70A1 protein functions at the intersection of two cellular pathways, where it is required in the stigma for the acceptance of compatible pollen in both Brassica and Arabidopsis and is negatively regulated by Brassica self-incompatibility. PMID:19789280

  5. Extracellular nucleotide inhibits cell proliferation and negatively regulates Toll-like receptor 4 signalling in human progenitor endothelial cells.

    PubMed

    Xiao, Zhilin; Yang, Mei; Fang, Li; Lv, Qingshan; He, Qing; Deng, Minjie; Liu, Xueting; Chen, Xiaobin; Chen, Meifang; Xie, Xiumei; Hu, Jinyue

    2012-07-01

    Extracellular nucleotides mediate a wide range of physiological effects by interacting with plasma membrane P2 purinergic receptors. P2 receptors are expressed in certain kinds of stem cells, and function to induce cytokine expression and to modulate cell proliferation. We have analysed the expression and the function of P2 receptors in human umbilical cord blood-derived EPCs (endothelial progenitor cells). EPCs expressed P2X4,6,7 and P2Y2,4,11,13,14 receptors and extracellular ATP inhibited EPCs proliferation. As in a previous study, EPCs expressed functional TLR4 (Toll-like receptor 4) and activation of TLR4 by LPS (lipopolysaccharide) evoked a pro-inflammatory immune response. When human EPCs were stimulated with LPS and nucleotides, ATP or UTP inhibited the expression of pro-inflammatory cytokines including MCP-1 (monocyte chemoattractant protein-1), IFNα (interferon α), TNFα (tumour necrosis factor α) and adhesion molecule VCAM-1 (vascular cell adhesion molecule 1) induced by LPS. ATP and UTP also down-regulated the gene expression of TLR4, CD14 and MyD88 (myeloid differentiation factor 88), a TLR adaptor molecule, and protein expression of CD14 and MyD88. Moreover, the phosphorylation of NF-κB (nuclear factor κB) p65 induced by TLR4 activation was inhibited partly by ATP or UTP at concentrations of 1-5 μM. These results suggest that extracellular nucleotides negatively regulate EPCs proliferation and TLR4 signalling.

  6. Arabidopsis RhoGDIs Are Critical for Cellular Homeostasis of Pollen Tubes.

    PubMed

    Feng, Qiang-Nan; Kang, Hui; Song, Shi-Jian; Ge, Fu-Rong; Zhang, Yu-Ling; Li, En; Li, Sha; Zhang, Yan

    2016-02-01

    Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth. © 2016 American Society of Plant Biologists. All Rights Reserved.

  7. Arabidopsis RhoGDIs Are Critical for Cellular Homeostasis of Pollen Tubes1[OPEN

    PubMed Central

    Feng, Qiang-Nan; Kang, Hui; Song, Shi-Jian; Ge, Fu-Rong; Zhang, Yu-Ling; Li, En; Li, Sha

    2016-01-01

    Rhos of plants (ROPs) play a key role in plant cell morphogenesis, especially in tip-growing pollen tubes and root hairs, by regulating an array of intracellular activities such as dynamic polymerization of actin microfilaments. ROPs are regulated by guanine nucleotide exchange factors (RopGEFs), GTPase activating proteins (RopGAPs), and guanine nucleotide dissociation inhibitors (RhoGDIs). RopGEFs and RopGAPs play evolutionarily conserved function in ROP signaling. By contrast, although plant RhoGDIs regulate the membrane extraction and cytoplasmic sequestration of ROPs, less clear are their positive roles in ROP signaling as do their yeast and metazoan counterparts. We report here that functional loss of all three Arabidopsis (Arabidopsis thaliana) GDIs (tri-gdi) significantly reduced male transmission due to impaired pollen tube growth in vitro and in vivo. We demonstrate that ROPs were ectopically activated at the lateral plasma membrane of the tri-gdi pollen tubes. However, total ROPs were reduced posttranslationally in the tri-gdi mutant, resulting in overall dampened ROP signaling. Indeed, a ROP5 mutant that was unable to interact with GDIs failed to induce growth, indicating the importance of the ROP-GDI interaction for ROP signaling. Functional loss of GDIs impaired cellular homeostasis, resulting in excess apical accumulation of wall components in pollen tubes, similar to that resulting from ectopic phosphatidylinositol 4,5-bisphosphate signaling. GDIs and phosphatidylinositol 4,5-bisphosphate may antagonistically coordinate to maintain cellular homeostasis during pollen tube growth. Our results thus demonstrate a more complex role of GDIs in ROP-mediated pollen tube growth. PMID:26662604

  8. Oscillatory Chloride Efflux at the Pollen Tube Apex Has a Role in Growth and Cell Volume Regulation and Is Targeted by Inositol 3,4,5,6-Tetrakisphosphate

    PubMed Central

    Zonia, Laura; Cordeiro, Sofia; Tupý, Jaroslav; Feijó, José A.

    2002-01-01

    overlaps the phase of cell elongation during the growth cycle. In summary, these investigations indicate that Cl− ion dynamics are an important component in the network of events that regulate pollen tube homeostasis and growth. PMID:6457666

  9. The MYB80 Transcription Factor Is Required for Pollen Development and the Regulation of Tapetal Programmed Cell Death in Arabidopsis thaliana[W][OA

    PubMed Central

    Phan, Huy Anh; Iacuone, Sylvana; Li, Song F.; Parish, Roger W.

    2011-01-01

    Arabidopsis thaliana MYB80 (formerly MYB103) is expressed in the tapetum and microspores between anther developmental stages 6 and 10. MYB80 encodes a MYB transcription factor that is essential for tapetal and pollen development. Using microarray analysis of anther mRNA, we identified 404 genes differentially expressed in the myb80 mutant. Employing the glucocorticoid receptor system, the expression of 79 genes was changed when MYB80 function was restored in the myb80 mutant following induction by dexamethasone. Thirty-two genes were analyzed using chromatin immunoprecipitation, and three were identified as direct targets of MYB80. The genes encode a glyoxal oxidase (GLOX1), a pectin methylesterase (VANGUARD1), and an A1 aspartic protease (UNDEAD). All three genes are expressed in the tapetum and microspores. Electrophoretic mobility shift assays confirmed that MYB80 binds to all three target promoters, with the preferential binding site containing the CCAACC motif. TUNEL assays showed that when UNDEAD expression was silenced using small interfering RNA, premature tapetal and pollen programmed cell death occurred, resembling the myb80 mutant phenotype. UNDEAD possesses a mitochondrial targeting signal and may hydrolyze an apoptosis-inducing protein(s) in mitochondria. The timing of tapetal programmed cell death is critical for pollen development, and the MYB80/UNDEAD system may regulate that timing. PMID:21673079

  10. The MYB80 transcription factor is required for pollen development and the regulation of tapetal programmed cell death in Arabidopsis thaliana.

    PubMed

    Phan, Huy Anh; Iacuone, Sylvana; Li, Song F; Parish, Roger W

    2011-06-01

    Arabidopsis thaliana MYB80 (formerly MYB103) is expressed in the tapetum and microspores between anther developmental stages 6 and 10. MYB80 encodes a MYB transcription factor that is essential for tapetal and pollen development. Using microarray analysis of anther mRNA, we identified 404 genes differentially expressed in the myb80 mutant. Employing the glucocorticoid receptor system, the expression of 79 genes was changed when MYB80 function was restored in the myb80 mutant following induction by dexamethasone. Thirty-two genes were analyzed using chromatin immunoprecipitation, and three were identified as direct targets of MYB80. The genes encode a glyoxal oxidase (GLOX1), a pectin methylesterase (VANGUARD1), and an A1 aspartic protease (UNDEAD). All three genes are expressed in the tapetum and microspores. Electrophoretic mobility shift assays confirmed that MYB80 binds to all three target promoters, with the preferential binding site containing the CCAACC motif. TUNEL assays showed that when UNDEAD expression was silenced using small interfering RNA, premature tapetal and pollen programmed cell death occurred, resembling the myb80 mutant phenotype. UNDEAD possesses a mitochondrial targeting signal and may hydrolyze an apoptosis-inducing protein(s) in mitochondria. The timing of tapetal programmed cell death is critical for pollen development, and the MYB80/UNDEAD system may regulate that timing.

  11. Trace concentrations of imazethapyr (IM) affect floral organs development and reproduction in Arabidopsis thaliana: IM-induced inhibition of key genes regulating anther and pollen biosynthesis.

    PubMed

    Qian, Haifeng; Li, Yali; Sun, Chongchong; Lavoie, Michel; Xie, Jun; Bai, Xiaocui; Fu, Zhengwei

    2015-01-01

    Understanding how herbicides affect plant reproduction and growth is critical to develop herbicide toxicity model and refine herbicide risk assessment. Although our knowledge of herbicides toxicity mechanisms at the physiological and molecular level in plant vegetative phase has increased substantially in the last decades, few studies have addressed the herbicide toxicity problematic on plant reproduction. Here, we determined the long-term (4-8 weeks) effect of a chiral herbicide, imazethapyr (IM), which has been increasingly used in plant crops, on floral organ development and reproduction in the model plant Arabidopsis thaliana. More specifically, we followed the effect of two IM enantiomers (R- and S-IM) on floral organ structure, seed production, pollen viability and the transcription of key genes involved in anther and pollen development. The results showed that IM strongly inhibited the transcripts of genes regulating A. thaliana tapetum development (DYT1: DYSFUNCTIONAL TAPETUM 1), tapetal differentiation and function (TDF1: TAPETAL DEVELOPMENT AND FUNCTION1), and pollen wall formation and developments (AMS: ABORTED MICROSPORES, MYB103: MYB DOMAIN PROTEIN 103, MS1: MALE STERILITY 1, MS2: MALE STERILITY 2). Since DYT1 positively regulates 33 genes involved in cell-wall modification (such as, TDF1, AMS, MYB103, MS1, MS2) that can catalyze the breakdown of polysaccharides to facilitate anther dehiscence, the consistent decrease in the transcription of these genes after IM exposure should hamper anther opening as observed under scanning electron microscopy. The toxicity of IM on anther opening further lead to a decrease in pollen production and pollen viability. Furthermore, long-term IM exposure increased the number of apurinic/apyrimidinic sites (AP sites) in the DNA of A. thaliana and also altered the DNA of A. thaliana offspring grown in IM-free soils. Toxicity of IM on floral organs development and reproduction was generally higher in the presence of the R

  12. An increase of oxidised nucleotides activates DNA damage checkpoint pathway that regulates post-embryonic development in Caenorhabditis elegans.

    PubMed

    Sanada, Yu; Zhang-Akiyama, Qiu-Mei

    2014-03-01

    8-Oxo-dGTP, an oxidised form of dGTP generated in the nucleotide pool, can be incorporated opposite adenine or cytosine in template DNA, which can in turn induce mutations. In this study, we identified a novel MutT homolog (NDX-2) of Caenorhabditis elegans that hydrolyzes 8-oxo-dGDP to 8-oxo-dGMP. In addition, we found that NDX-1, NDX-2 and NDX-4 proteins have 8-oxo-GTPase or 8-oxo-GDPase activity. The sensitivity of ndx-2 knockdown C. elegans worms to methyl viologen and menadione bisulphite was increased compared with that of control worms. This sensitivity was rescued by depletion of chk-2 and clk-2, suggesting that growth of the worms is regulated by the checkpoint pathway in response to the accumulation of oxidised nucleotides. Moreover, we found that the sensitivity to menadione bisulphite of ndx-1 and ndx-2-double knockdown worms was enhanced by elimination of XPA-1, a factor involved in nucleotide excision repair. The rescue effect by depletion of chk-2 and clk-2 was limited in the xpa-1 mutant, suggesting that the chk-2 and clk-2 checkpoint pathway is partially linked to the function of XPA-1.

  13. A nucleotide-regulated Cl-/OH- anion exchanger in endoplasmic reticulum-enriched pig pancreatic microsomes.

    PubMed

    Bégault, B; Edelman, A

    1993-03-14

    The anion conductive pathways in preparations of endoplasmic reticulum (ER)-enriched microsomes from pig pancreas were investigated. The rate of swelling induced by cation ionophores (nigericin (nig) and/or valinomycin (val)) was measured in iso-osmotic solutions by light scattering, in the presence or absence of an inward Cl- and/or pH gradients. The rate of swelling in the presence of the inward Cl- gradient and ionophores was faster than that of controls. Low pH did not change the swelling rate in the presence of valinomycin, but it increased it in the presence of nigericin. When the Cl- gradient was abolished, valinomycin plus the pH gradient increased the rate of swelling, and this was further enhanced by nigericin. Anion transport inhibitors reduced the swelling rate. The nigericin-induced swelling was stimulated by ATP and GTP. The non-hydrolysable analogues, adenosine 5'-[beta,gamma-methylene]triphosphate, guanosine 5'-[beta-thio]triphosphate and guanosine 5'-[beta-thio]diphosphate, increased the rate of swelling, whereas adenosine 5'-[gamma-thio]triphosphate inhibited it. ADP, CTP and UTP had no effect. These data suggest the presence of a Cl-/OH- exchanger and a Cl- conductance in microsomes. They indicate that nucleotides may regulate the Cl-/OH- exchanger. Nucleotides do not need to be hydrolyzed but phosphorylation may occur to counter-balance the nucleotide-induced stimulation.

  14. The guanine nucleotide exchange factor Vav3 regulates differentiation of progenitor cells in the developing mouse retina.

    PubMed

    Luft, Veronika; Reinhard, Jacqueline; Shibuya, Masabumi; Fischer, Klaus D; Faissner, Andreas

    2015-02-01

    The seven main cell types in the mammalian retina arise from multipotent retinal progenitor cells, a process that is tightly regulated by intrinsic and extrinsic signals. However, the molecular mechanisms that control proliferation, differentiation and cell-fate decisions of retinal progenitor cells are not fully understood yet. Here, we report that the guanine nucleotide exchange factor Vav3, a regulator of Rho-GTPases, is involved in retinal development. We demonstrate that Vav3 is expressed in the mouse retina during the embryonic period. In order to study the role of Vav3 in the developing retina, we generate Vav3-deficient mice. The loss of Vav3 results in an accelerated differentiation of retinal ganglion cells and cone photoreceptors during early and late embryonic development. We provide evidence that more retinal progenitor cells express the late progenitor marker Sox9 in Vav3-deficient mice than in wild-types. This premature differentiation is compensated during the postnatal period and late-born cell types such as bipolar cells and Müller glia display normal numbers. Taken together, our data imply that Vav3 is a regulator of retinal progenitor cell differentiation, thus highlighting a novel role for guanine nucleotide exchange factors in retinogenesis.

  15. Arabidopsis MALE STERILITY1 encodes a PHD-type transcription factor and regulates pollen and tapetum development.

    PubMed

    Ito, Takuya; Nagata, Noriko; Yoshiba, Yoshu; Ohme-Takagi, Masaru; Ma, Hong; Shinozaki, Kazuo

    2007-11-01

    The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper-like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper-like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants.

  16. Effects of antihistamine on up-regulation of histamine H1 receptor mRNA in the nasal mucosa of patients with pollinosis induced by controlled cedar pollen challenge in an environmental exposure unit.

    PubMed

    Kitamura, Yoshiaki; Nakagawa, Hideyuki; Fujii, Tatsuya; Sakoda, Takema; Enomoto, Tadao; Mizuguchi, Hiroyuki; Fukui, Hiroyuki; Takeda, Noriaki

    2015-11-01

    In the present study, we examined the effects of antihistamine on the up-regulation of H1R mRNA in the nasal mucosa of patients with pollinosis induced by controlled exposure to pollen using an environmental exposure unit. Out of 20 patients, we designated 14 responders, whose levels of H1R mRNA in the nasal mucosa were increased after the first pollen exposure and excluded 6 non-responders. Accordingly, the first exposure to pollen without treatment significantly induced both nasal symptoms and the up-regulation of H1R mRNA in the nasal mucosa of the responders. Subsequently, prophylactic administration of antihistamine prior to the second pollen exposure significantly inhibited both of the above effects in the responders. Moreover, the nasal expression of H1R mRNA before the second pollen exposure in the responders pretreated with antihistamine was significantly decreased, as compared with that before the first pollen exposure without treatment. These findings suggest that antihistamines suppressed histamine-induced transcriptional activation of H1R gene in the nasal mucosa, in addition to their blocking effect against histamine on H1R, resulting in a decrease of nasal symptoms. These findings further suggest that by their inverse agonistic activity, antihistamines suppress the basal transcription of nasal H1R in the absence of histamine in responders.

  17. CYCLIN-DEPENDENT KINASE G1 is associated with the spliceosome to regulate CALLOSE SYNTHASE5 splicing and pollen wall formation in Arabidopsis.

    PubMed

    Huang, Xue-Yong; Niu, Jin; Sun, Ming-Xi; Zhu, Jun; Gao, Ju-Fang; Yang, Jun; Zhou, Que; Yang, Zhong-Nan

    2013-02-01

    Arabidopsis thaliana CYCLIN-DEPEDENT KINASE G1 (CDKG1) belongs to the family of cyclin-dependent protein kinases that were originally characterized as cell cycle regulators in eukaryotes. Here, we report that CDKG1 regulates pre-mRNA splicing of CALLOSE SYNTHASE5 (CalS5) and, therefore, pollen wall formation. The knockout mutant cdkg1 exhibits reduced male fertility with impaired callose synthesis and abnormal pollen wall formation. The sixth intron in CalS5 pre-mRNA, a rare type of intron with a GC 5' splice site, is abnormally spliced in cdkg1. RNA immunoprecipitation analysis suggests that CDKG1 is associated with this intron. CDKG1 contains N-terminal Ser/Arg (RS) motifs and interacts with splicing factor Arginine/Serine-Rich Zinc Knuckle-Containing Protein33 (RSZ33) through its RS region to regulate proper splicing. CDKG1 and RS-containing Zinc Finger Protein22 (SRZ22), a splicing factor interacting with RSZ33 and U1 small nuclear ribonucleoprotein particle (snRNP) component U1-70k, colocalize in nuclear speckles and reside in the same complex. We propose that CDKG1 is recruited to U1 snRNP through RSZ33 to facilitate the splicing of the sixth intron of CalS5.

  18. A Steric-inhibition model for regulation of nucleotide exchange via the Dock180 family of GEFs.

    PubMed

    Lu, Mingjian; Kinchen, Jason M; Rossman, Kent L; Grimsley, Cynthia; Hall, Matthew; Sondek, John; Hengartner, Michael O; Yajnik, Vijay; Ravichandran, Kodi S

    2005-02-22

    CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

  19. A secondary structural transition in the C-helix promotes gating of cyclic nucleotide-regulated ion channels.

    PubMed

    Puljung, Michael C; Zagotta, William N

    2013-05-03

    Cyclic nucleotide-regulated ion channels bind second messengers like cAMP to a C-terminal domain, consisting of a β-roll, followed by two α-helices (B- and C-helices). We monitored the cAMP-dependent changes in the structure of the C-helix of a C-terminal fragment of HCN2 channels using transition metal ion FRET between fluorophores on the C-helix and metal ions bound between histidine pairs on the same helix. cAMP induced a change in the dimensions of the C-helix and an increase in the metal binding affinity of the histidine pair. cAMP also caused an increase in the distance between a fluorophore on the C-helix and metal ions bound to the B-helix. Stabilizing the C-helix of intact CNGA1 channels by metal binding to a pair of histidines promoted channel opening. These data suggest that ordering of the C-helix is part of the gating conformational change in cyclic nucleotide-regulated channels.

  20. Quantitative determination of binding of ISWI to nucleosomes and DNA shows allosteric regulation of DNA binding by nucleotides.

    PubMed

    Al-Ani, Gada; Briggs, Koan; Malik, Shuja Shafi; Conner, Michael; Azuma, Yoshiaki; Fischer, Christopher J

    2014-07-15

    The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM(2). Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP.

  1. Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration

    PubMed Central

    Marei, Hadir; Carpy, Alejandro; Woroniuk, Anna; Vennin, Claire; White, Gavin; Timpson, Paul; Macek, Boris; Malliri, Angeliki

    2016-01-01

    The small GTPase Rac1 has been implicated in the formation and dissemination of tumours. Upon activation by guanine nucleotide exchange factors (GEFs), Rac1 associates with a variety of proteins in the cell thereby regulating various functions, including cell migration. However, activation of Rac1 can lead to opposing migratory phenotypes raising the possibility of exacerbating tumour progression when targeting Rac1 in a clinical setting. This calls for the identification of factors that influence Rac1-driven cell motility. Here we show that Tiam1 and P-Rex1, two Rac GEFs, promote Rac1 anti- and pro-migratory signalling cascades, respectively, through regulating the Rac1 interactome. In particular, we demonstrate that P-Rex1 stimulates migration through enhancing the interaction between Rac1 and the actin-remodelling protein flightless-1 homologue, to modulate cell contraction in a RhoA-ROCK-independent manner. PMID:26887924

  2. Regulation of global genome nucleotide excision repair by SIRT1 through xeroderma pigmentosum C

    PubMed Central

    Ming, Mei; Shea, Christopher R.; Guo, Xiumei; Li, Xiaoling; Soltani, Keyoumars; Han, Weinong; He, Yu-Ying

    2010-01-01

    Disruption of the nucleotide excision repair (NER) pathway by mutations can cause xeroderma pigmentosum, a syndrome predisposing affected individuals to development of skin cancer. The xeroderma pigmentosum C (XPC) protein is essential for initiating global genome NER by recognizing the DNA lesion and recruiting downstream factors. Here we show that inhibition of the deacetylase and longevity factor SIRT1 impairs global genome NER through suppressing the transcription of XPC in a SIRT1 deacetylase-dependent manner. SIRT1 enhances XPC expression by reducing AKT-dependent nuclear localization of the transcription repressor of XPC. Finally, we show that SIRT1 levels are significantly reduced in human skin tumors from Caucasian patients, a population at highest risk. These findings suggest that SIRT1 acts as a tumor suppressor through its role in DNA repair. PMID:21149730

  3. UVSSA and USP7: new players regulating transcription-coupled nucleotide excision repair in human cells

    PubMed Central

    2012-01-01

    Transcription-coupled nucleotide excision repair (TC-NER) specifically removes DNA damage located in actively transcribed genes. Defects in TC-NER are associated with several human disorders, including Cockayne syndrome (CS) and ultraviolet (UV)-sensitive syndrome (UVSS). Using exome sequencing, and genetic and proteomic approaches, three recent studies have identified mutations in the UVSSA gene as being responsible for UVSS-A. These findings suggest a new mechanistic model involving UV-stimulated scaffold protein A (UVSSA) and the ubiquitin-specific protease 7 (USP7) in the fate of stalled RNA polymerase II during TC-NER, and provide insights into the diverse clinical features of CS and UVSS. PMID:22621766

  4. Nucleotide regulation of paracellular Cl- permeability in natural rabbit airway epithelium.

    PubMed

    Poulsen, Asser Nyander; Klausen, Thomas Levin; Pedersen, Peter Steen; Willumsen, Niels Johannes; Frederiksen, Ole

    2006-05-01

    In this study, we demonstrate a novel regulatory mechanism by which mucosal nucleotides via P2Y receptors decrease paracellular Cl(-) ion permeability in natural rabbit airway epithelium (in addition to a decrease in active Na(+) absorption). In contrast to primary cultures, the natural airway epithelium is a low-resistance epithelium, and an equivalent circuit model predicts that changes of more than approximately 12% in transepithelial conductance (G (t)) must include an effect on paracellular conductance (G (s)). Mucosal P2Y receptor stimulation with uridine triphosphate (UTP; 200 microM) decreased G (t) by up to 50% (average, 24%) and simultaneously decreased the paracellular Cl(-) permeability (mucosa-to-serosa Cl(-) flux) by 16%, but had no effect on mannitol permeability. The G (t) response to UTP was mimicked and attenuated by ionomycin (1 microM), suggesting a dependence on Ca(2+) (i). Amiloride (100 microM) and hyperosmolarity (+75 mM mannitol) also decreased G (t), indicating a role of cell shrinkage. Elevation of cAMP with forskolin (8 microM) or isoproterenol (10 microM) increased G (t) by 55 and 32%, and forskolin increased paracellular Cl(-) permeability by 37% without affecting mannitol permeability. The opposite effects of Ca(2+) (i) and cAMP on G (t) suggest an autocrine nucleotide signaling sequence where P2Y-dependent decrease in passive, paracellular Cl(-) transport is succeeded by a reversion of this effect due to P1-receptor-stimulated cAMP formation by adenosine originating from a time-dependent breakdown of mucosal ATP.

  5. Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

    PubMed

    Nicholas, Matthew P; Berger, Florian; Rao, Lu; Brenner, Sibylle; Cho, Carol; Gennerich, Arne

    2015-05-19

    Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

  6. Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP.

    PubMed

    Serina, L; Blondin, C; Krin, E; Sismeiro, O; Danchin, A; Sakamoto, H; Gilles, A M; Bârzu, O

    1995-04-18

    The pyrH gene, encoding UMP-kinase from Escherichia coli, was cloned using as a genetic probe the property of the carAB operon to be controlled for its expression by the concentration of cytoplasmic UTP. The open reading frame of the pyrH gene of 723 bp was found to be identical to that of the smbA gene [Yamanaka, K., et al. (1992) J. Bacteriol. 174, 7517-7526], previously described as being involved in chromosome partitioning in E. coli. The bacterial UMP-kinase did not display significant sequence similarity to known nucleoside monophosphate kinases. On the contrary, it exhibited similarity with three families of enzymes including aspartokinases, glutamate kinases, and Pseudomonas aeruginosa carbamate kinase. UMP-kinase overproduced in E. coli was purified to homogeneity and analyzed for its structural and catalytic properties. The protein consists of six identical subunits, each of 240 amino acid residues (the N-terminal methionine residue is missing in the expressed protein). Upon excitation at 295 nm, the bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 332 nm which indicates that the single tryptophan residue of the protein (Trp119) is located in a hydrophobic environment. Like other enzymes involved in the de novo synthesis of pyrimidine nucleotides, UMP-kinase of E. coli is subject to regulation by nucleotides: GTP is an allosteric activator, whereas UTP serves as an allosteric inhibitor. UTP and UDP, but none of the other nucleotides tested such as GTP, ATP, and UMP, enhanced the fluorescence of the protein. The sigmoidal shape of the dose-response curve indicated cooperativity in binding of UTP and UDP.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Quantitative Nucleotide Level Analysis of Regulation of Translation in Response to Depolarization of Cultured Neural Cells

    PubMed Central

    Dalal, Jasbir S.; Yang, Chengran; Sapkota, Darshan; Lake, Allison M.; O'Brien, David R.; Dougherty, Joseph D.

    2017-01-01

    Studies on regulation of gene expression have contributed substantially to understanding mechanisms for the long-term activity-dependent alterations in neural connectivity that are thought to mediate learning and memory. Most of these studies, however, have focused on the regulation of mRNA transcription. Here, we utilized high-throughput sequencing coupled with ribosome footprinting to globally characterize the regulation of translation in primary mixed neuronal-glial cultures in response to sustained depolarization. We identified substantial and complex regulation of translation, with many transcripts demonstrating changes in ribosomal occupancy independent of transcriptional changes. We also examined sequence-based mechanisms that might regulate changes in translation in response to depolarization. We found that these are partially mediated by features in the mRNA sequence—notably upstream open reading frames and secondary structure in the 5′ untranslated region—both of which predict downregulation in response to depolarization. Translationally regulated transcripts are also more likely to be targets of FMRP and include genes implicated in autism in humans. Our findings support the idea that control of mRNA translation plays an important role in response to neural activity across the genome. PMID:28190998

  8. Gametophytic Pollen Tube Guidance: Attractant Peptides, Gametic Controls, and Receptors.

    PubMed

    Higashiyama, Tetsuya; Yang, Wei-Cai

    2017-01-01

    Pollen tube guidance in flowering plants is a unique and critical process for successful sexual reproduction. The pollen tube that grows from pollen, which is the male gametophyte, precisely navigates to the embryo sac, which is the female gametophyte, within the pistil. Recent advances have clarified the molecular framework of gametophytic pollen tube guidance. Multiple species-specific attractant peptides are secreted from synergid cells, the proper development and function of which are regulated by female gametes. Multiple receptor-like kinases on the pollen tube tip are involved in sensing species-specific attractant peptides. In this Update article, recent progress in our understanding of the mechanism of gametophytic pollen tube guidance is reviewed, including attraction by synergid cells, control of pollen tube guidance by female gametes, and directional growth of the pollen tube by directional cue sensing. Future directions in the study of pollen tube guidance also are discussed.

  9. Optimized method for growing in vitro Arabidopsis thaliana pollen tubes.

    PubMed

    Borassi, Cecilia; Di Giorgio, Juliana Pérez; Scarpin, María R; Muschietti, Jorge; Estevez, José M

    2015-01-01

    Pollen tubes elongate by tip growth toward the ovule to deliver the sperm cells during fertilization. Since pollen tubes from several species can be grown in vitro maintaining their polarity, pollen tube growth is a suitable model system to study cell polarity and tip growth. A. thaliana pollen tubes germinated in vitro for 6 h can reach up to 800 μm. By studying the phenotype of mutants of T-DNA insertion lines, genes involved in pollen tube growth can be identified. Moreover, components involved in the regulation of pollen tube growth such as calcium ions and reactive oxygen species (ROS) can be analyzed.

  10. Adenine Nucleotide Translocator as a Regulator of Mitochondrial Function: Implication in the Pathogenesis of Metabolic Syndrome

    PubMed Central

    Kim, Eun Hee; Koh, Eun Hee; Park, Joong-Yeol

    2010-01-01

    Mitochondria play key roles in energy production and intracellular reactive oxygen species (ROS) generation. Lines of evidence have shown that mitochondrial dysfunction contributes to the development of metabolic syndrome. The causes of mitochondrial dysfunction are complex, but overnutrition and sedentary living are among the best known causes of mitochondrial dysfunction. ATP synthesized in the mitochondria is exchanged for cytosolic ADP by adenine nucleotide translocator (ANT) to provide a continuous supply of ADP to mitochondria. We recently found that ANT function is essential for peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α)'s action on endothelial cells. PGC-1α is a transcriptional coactivator of nuclear receptors, playing an important role in fatty acid oxidation and mitochondrial biogenesis. Recent studies have shown that PGC-1α decreases intracellular ROS generation by increasing the expression of antioxidant genes. In our study, PGC-1α reduced cell apoptosis and ROS generation in endothelial cells by increasing ATP/ADP translocase activity of ANT and ANT1 expression. Here we review the role of ANT in maintaining proper mitochondrial function, and possible role of ANT dysfunction in the pathogenesis of metabolic syndrome. PMID:20617074

  11. Single-nucleotide polymorphisms and DNA methylation markers associated with central obesity and regulation of body weight.

    PubMed

    Goni, Leticia; Milagro, Fermín I; Cuervo, Marta; Martínez, J Alfredo

    2014-11-01

    Visceral fat is strongly associated with the development of specific obesity-related metabolic alterations. Genetic and epigenetic mechanisms seem to be involved in the development of obesity and visceral adiposity. The aims of this review are to identify the single-nucleotide polymorphisms related to central obesity and to summarize the main findings on DNA methylation and obesity. A search of the MEDLINE database was conducted to identify genome-wide association studies, meta-analyses of genome-wide association studies, and gene-diet interaction studies related to central obesity, and, in addition, studies that analyzed DNA methylation in relation to body weight regulation. A total of 8 genome-wide association studies and 9 meta-analyses of genome-wide association studies reported numerous single-nucleotide polymorphisms to be associated with central obesity. Ten studies analyzed gene-diet interactions and central obesity, while 2 epigenome-wide association studies analyzed DNA methylation patterns and obesity. Nine studies investigated the relationship between DNA methylation and weight loss, excess body weight, or adiposity outcomes. Given the development of new sequencing and omics technologies, significantly more knowledge on genomics and epigenomics of obesity and body fat distribution will emerge in the near future.

  12. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    SciTech Connect

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M. )

    1989-05-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using {sup 125}I-labeled melatonin ({sup 125}I-Mel), a potent melatonin agonist. {sup 125}I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K{sub d} of 2.3 {plus minus} 1.0 {times} 10{sup {minus}11} M and 2.06 {plus minus} 0.43 {times} 10{sup {minus}10} M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5{prime}-({gamma}-thio)triphosphate (GTP({gamma}S)), significantly reduced the number of high-affinity receptors and increased the dissociation rate of {sup 125}I-Mel from its receptor. Furthermore, GTP({gamma}S) treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of {sup 125}I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M{sub r} > 400,000 and M{sub r} ca. 110,000. This elution profile was markedly altered by pretreatment with GTP({gamma}S) before solubilization; only the M{sub r} 110,000 peak was present in GTP({gamma}S)-pretreated membranes. The results strongly suggest that {sup 125}I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000.

  13. STRUCTURAL BASIS FOR ALLOSTERIC REGULATION OF HUMAN RIBONUCLEOTIDE REDUCTASE BY NUCLEOTIDE-INDUCED OLIGOMERIZATION

    PubMed Central

    Fairman, James Wesley; Wijerathna, Sanath Ranjan; Ahmad, Md. Faiz; Xu, Hai; Nakano, Ryo; Jha, Shalini; Prendergast, Jay; Welin, Martin; Flodin, Susanne; Roos, Annette; Nordlund, Pär; Li, Zongli; Walz, Thomas; Dealwis, Chris Godfrey

    2011-01-01

    Ribonucleotide reductase (RR) is an αnβn (RR1●RR2) complex that maintains balanced dNTP pools by reducing ribonucleoside diphosphates to deoxyribonucleoside diphosphates. RR1 is the catalytic subunit and RR2 houses the free radical required for catalysis. RR is allosterically regulated by its activator ATP and its inhibitor dATP, which regulate RR activity by inducing oligomerization of RR1. Here, we report the first X-ray structures of human RR1 bound to TTP-only, dATP-only, TTP●GDP, TTP●ATP, and TTP●dATP. These structures provide insights into ATP/dATP regulation of RR. At physiological dATP concentrations, RR1 forms inactive hexamers. We determined the first X-ray structure of the RR1●dATP hexamer and used single-particle electron microscopy to visualize the α6●ββ’ 1●dATP holo complex. Site-directed mutagenesis and functional assays confirm that hexamerization is a prerequisite for inhibition by dATP. Our data provide an elegant mechanism for regulating RR activity by dATP-induced oligomerization. PMID:21336276

  14. Quantitative Determination of Binding of ISWI to Nucleosomes and DNA Shows Allosteric Regulation of DNA Binding by Nucleotides

    PubMed Central

    2015-01-01

    The regulation of chromatin structure is controlled by a family of molecular motors called chromatin remodelers. The ability of these enzymes to remodel chromatin structure is dependent on their ability to couple ATP binding and hydrolysis into the mechanical work that drives nucleosome repositioning. The necessary first step in determining how these essential enzymes perform this function is to characterize both how they bind nucleosomes and how this interaction is regulated by ATP binding and hydrolysis. With this goal in mind, we monitored the interaction of the chromatin remodeler ISWI with fluorophore-labeled nucleosomes and DNA through associated changes in fluorescence anisotropy of the fluorophore upon binding of ISWI to these substrates. We determined that one ISWI molecule binds to a 20 bp double-stranded DNA substrate with an affinity of 18 ± 2 nM. In contrast, two ISWI molecules can bind to the core nucleosome with short linker DNA with stoichiometric macroscopic equilibrium constants: 1/β1 = 1.3 ± 0.6 nM, and 1/β2 = 13 ± 7 nM2. Furthermore, to improve our understanding of the mechanism of DNA translocation by ISWI, and hence nucleosome repositioning, we determined the effect of nucleotide analogues on substrate binding by ISWI. While the affinity of ISWI for the nucleosome substrate with short lengths of flanking DNA was not affected by the presence of nucleotides, the affinity of ISWI for the DNA substrate is weakened in the presence of nonhydrolyzable ATP analogues but not by ADP. PMID:24898734

  15. The speed of mitochondrial movement is regulated by the cytoskeleton and myosin in Picea wilsonii pollen tubes.

    PubMed

    Zheng, Maozhong; Wang, Qinli; Teng, Yan; Wang, Xiaohua; Wang, Feng; Chen, Tong; Samaj, Jozef; Lin, Jinxing; Logan, David C

    2010-03-01

    Strategic control of mitochondrial movements and cellular distribution is essential for correct cell function and survival. However, despite being a vital process, mitochondrial movement in plant cells is a poorly documented phenomenon. To investigate the roles of actin filaments and microtubules on mitochondrial movements, Picea wilsonii pollen tubes were treated with two microtubule-disrupting drugs, two actin-disrupting drugs and a myosin inhibitor. Following these treatments, mitochondrial movements were characterized by multiangle evanescent wave microscopy and laser-scanning confocal microscopy. The results showed that individual mitochondria underwent three classes of linear movement: high-speed movement (instantaneous velocities >5.0 microm/s), low-speed movement (instantaneous velocities <5.0 microm/s) and variable-speed movement (instantaneous velocities ranging from 0.16 to 10.35 microm/s). 10 nM latrunculin B induced fragmentation of actin filaments and completely inhibited mitochondrial vectorial movement. Jasplakinolide treatment induced a 28% reduction in chondriome motility, and dramatically inhibition of high-speed and variable-speed movements. Treatment with 2,3-butanedione 2-monoxime caused a 61% reduction of chondriome motility, and the complete inhibition of high-speed and low-speed movements. In contrast to actin-disrupting drugs, microtubule-disrupting drugs caused mild effects on mitochondrial movement. Taxol increased the speed of mitochondrial movement in cortical cytoplasm. Oryzalin induced curved mitochondrial trajectories with similar velocities as in the control pollen tubes. These results suggest that mitochondrial movement at low speeds in pollen tubes is driven by myosin, while high-speed and variable-speed movements are powered both by actin filament dynamics and myosin. In addition, microtubule dynamics has profound effects on mitochondrial velocity, trajectory and positioning via its role in directing the arrangement of actin

  16. 7 CFR 201.78 - Pollen control for hybrids.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... 7 Agriculture 3 2013-01-01 2013-01-01 false Pollen control for hybrids. 201.78 Section 201.78... REGULATIONS Additional Requirements for the Certification of Plant Materials of Certain Crops § 201.78 Pollen... branches, or any combination thereof, shedding pollen. (c) Sorghum. Shedders in the seed parent, at any...

  17. 7 CFR 201.78 - Pollen control for hybrids.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... 7 Agriculture 3 2014-01-01 2014-01-01 false Pollen control for hybrids. 201.78 Section 201.78... REGULATIONS Additional Requirements for the Certification of Plant Materials of Certain Crops § 201.78 Pollen... branches, or any combination thereof, shedding pollen. (c) Sorghum. Shedders in the seed parent, at any...

  18. 7 CFR 201.78 - Pollen control for hybrids.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 3 2011-01-01 2011-01-01 false Pollen control for hybrids. 201.78 Section 201.78... REGULATIONS Additional Requirements for the Certification of Plant Materials of Certain Crops § 201.78 Pollen... branches, or any combination thereof, shedding pollen. (c) Sorghum. Shedders in the seed parent, at any...

  19. 7 CFR 201.78 - Pollen control for hybrids.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... 7 Agriculture 3 2012-01-01 2012-01-01 false Pollen control for hybrids. 201.78 Section 201.78... REGULATIONS Additional Requirements for the Certification of Plant Materials of Certain Crops § 201.78 Pollen... branches, or any combination thereof, shedding pollen. (c) Sorghum. Shedders in the seed parent, at any...

  20. 7 CFR 201.78 - Pollen control for hybrids.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 3 2010-01-01 2010-01-01 false Pollen control for hybrids. 201.78 Section 201.78... REGULATIONS Additional Requirements for the Certification of Plant Materials of Certain Crops § 201.78 Pollen... branches, or any combination thereof, shedding pollen. (c) Sorghum. Shedders in the seed parent, at any...

  1. Single Nucleotide Polymorphisms Can Create Alternative Polyadenylation Signals and Affect Gene Expression through Loss of MicroRNA-Regulation

    PubMed Central

    Thomas, Laurent F.; Sætrom, Pål

    2012-01-01

    Alternative polyadenylation (APA) can for example occur when a protein-coding gene has several polyadenylation (polyA) signals in its last exon, resulting in messenger RNAs (mRNAs) with different 3′ untranslated region (UTR) lengths. Different 3′UTR lengths can give different microRNA (miRNA) regulation such that shortened transcripts have increased expression. The APA process is part of human cells' natural regulatory processes, but APA also seems to play an important role in many human diseases. Although altered APA in disease can have many causes, we reasoned that mutations in DNA elements that are important for the polyA process, such as the polyA signal and the downstream GU-rich region, can be one important mechanism. To test this hypothesis, we identified single nucleotide polymorphisms (SNPs) that can create or disrupt APA signals (APA-SNPs). By using a data-integrative approach, we show that APA-SNPs can affect 3′UTR length, miRNA regulation, and mRNA expression—both between homozygote individuals and within heterozygote individuals. Furthermore, we show that a significant fraction of the alleles that cause APA are strongly and positively linked with alleles found by genome-wide studies to be associated with disease. Our results confirm that APA-SNPs can give altered gene regulation and that APA alleles that give shortened transcripts and increased gene expression can be important hereditary causes for disease. PMID:22915998

  2. Nucleotides and nucleoside signaling in the regulation of the epithelium to mesenchymal transition (EMT).

    PubMed

    Martínez-Ramírez, A S; Díaz-Muñoz, M; Butanda-Ochoa, A; Vázquez-Cuevas, F G

    2017-03-01

    The epithelium-mesenchymal transition (EMT) is an important process of cell plasticity, consisting in the loss of epithelial identity and the gain of mesenchymal characteristics through the coordinated activity of a highly regulated informational program. Although it was originally described in the embryonic development, an important body of information supports its role in pathology, mainly in cancerous and fibrotic processes. The purinergic system of inter-cellular communication, mainly based in ATP and adenosine acting throughout their specific receptors, has emerged as a potent regulator of the EMT in several pathological entities. In this context, cellular signaling associated to purines is opening the understanding of a new element in the complex regulatory network of this phenotypical differentiation process. In this review, we have summarized recent information about the role of ATP and adenosine in EMT, as a growing field with high therapeutic potential.

  3. Nucleotide second messenger-mediated regulation of a muralytic enzyme in Streptomyces.

    PubMed

    St-Onge, Renée J; Haiser, Henry J; Yousef, Mary R; Sherwood, Emma; Tschowri, Natalia; Al-Bassam, Mahmoud; Elliot, Marie A

    2015-05-01

    Peptidoglycan degradative enzymes have important roles at many stages during the bacterial life cycle, and it is critical that these enzymes be stringently regulated to avoid compromising the integrity of the cell wall. How this regulation is exerted is of considerable interest: promoter-based control and protein-protein interactions are known to be employed; however, other regulatory mechanisms are almost certainly involved. In the actinobacteria, a class of muralytic enzymes - the 'resuscitation-promoting factors' (Rpfs) - orchestrates the resuscitation of dormant cells. In this study, we have taken a holistic approach to exploring the mechanisms governing RpfA function using the model bacterium Streptomyces coelicolor and have uncovered unprecedented multilevel regulation that is coordinated by three second messengers. Our studies show that RpfA is subject to transcriptional control by the cyclic AMP receptor protein, riboswitch-mediated transcription attenuation in response to cyclic di-AMP, and growth stage-dependent proteolysis in response to ppGpp accumulation. Furthermore, our results suggest that these control mechanisms are likely applicable to cell wall lytic enzymes in other bacteria.

  4. Anti-proliferative activity of L-651,582 correlates with calcium-mediated regulation of nucleotide metabolism at phosphoribosyl pyrophosphate synthetase

    SciTech Connect

    Hupe, D.J.; Behrens, N.D.; Boltz, R. )

    1990-09-01

    L-651,582, 5-amino-(4-(4-chlorobenzoyl)-3,5-dichlorobenzyl)-1, 2,3-triazole-4-carboxamide, is an antiproliferative and antiparasitic agent which inhibits nucleotide metabolism in mammalian cells. The drug equivalently inhibited 3H-hypoxanthine, 14C-adenine, and 14C-formate incorporation into nucleotide pools in Madin-Darby bovine kidney (MDBK) cells, suggesting depletion of the supply of phosphoribosyl pyrophosphate, (PRPP), required for each of these independent pathways. Inhibition of nucleotide metabolism correlated with inhibition of proliferation for three cell types with differing sensitivities toward the drug. L-651,582 inhibited incorporation of 3H-hypoxanthine into nucleotide pools with either glucose, uridine, or ribose as carbon source suggesting a block at PRPP synthetase, rather than a block in a pathway supplying ribose-5-phosphate. PRPP synthetase was not inhibited directly by the compound, indicating regulation of the enzyme in intact cells. Drug treatment did not kill cells but reduced the fraction of cells in S and G2/M while increasing the population in G1. Inhibition of uptake of 45Ca was demonstrated at concentrations identical to those required for inhibition of nucleotide metabolism or proliferation. Inhibition of cellular PRPP biosynthesis rates were also observed using EGTA to lower calcium levels. These data suggest a previously unrecognized link between calcium entry, the regulation of nucleotide biosynthesis at PRPP synthetase, and the rate of proliferation of mammalian cells.

  5. Role of global regulators and nucleotide metabolism in antibiotic tolerance in Escherichia coli.

    PubMed

    Hansen, Sonja; Lewis, Kim; Vulić, Marin

    2008-08-01

    Bacterial populations produce a small number of persister cells that exhibit multidrug tolerance. Persister cells are largely responsible for the antibiotic recalcitrance of biofilm infections. The mechanism of persister cell formation largely remains unknown due to the challenges in identifying persister genes. We screened an ordered comprehensive library of 3,985 Escherichia coli knockout strains to identify mutants with altered antibiotic tolerance. Stationary-state cultures in 96-well plates were exposed to ofloxacin at a concentration which allows only tolerant persister cells to survive. The persister cell level of each culture was determined. A total of 150 mutants with decreased persistence were identified in the initial screen, and subsequent validation confirmed that neither the growth rate nor the ofloxacin MIC was affected for 10 of them. The genes affected in these strains were dnaJ and dnaK (chaperones), apaH (diadenosine tetraphosphatase), surA (peptidyl-prolyl cis-trans isomerase), fis and hns (global regulators), hnr (response regulator of RpoS), dksA (transcriptional regulator of rRNA transcription), ygfA (5-formyl-tetrahydrofolate cyclo-ligase), and yigB (flavin mononucleotide [FMN] phosphatase). The prominent presence of global regulators among these strains pointed to the likely redundancy of persister cell formation mechanisms: the elimination of a regulator controlling several redundant persister genes would be expected to produce a phenotype. This observation is consistent with previous findings for a possible role of redundant genes such as toxin/antitoxin modules in persister cell formation. ygfA and yigB were of special interest. The mammalian homolog of YgfA (methenyltetrahydrofolate synthetase) catalyzes the conversion of 5-formyl-tetrahydrofolate (THF) into the rapidly degraded 5,10-methenyl-THF, depleting the folate pool. The YigB protein is a phosphatase of FMN which would deplete the pool of this cofactor. Stochastic overexpression

  6. Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase

    PubMed Central

    Yu, Guang-Hui; Zou, Jie; Feng, Jing; Peng, Xiong-Bo; Wu, Ju-You; Wu, Ying-Liang; Palanivelu, Ravishankar; Sun, Meng-Xiang

    2014-01-01

    γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca2+-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca2+ increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca2+-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca2+-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes. PMID:24799560

  7. Oscillatory Increases in Alkalinity Anticipate Growth and May Regulate Actin Dynamics in Pollen Tubes of Lily[W][OA

    PubMed Central

    Lovy-Wheeler, Alenka; Kunkel, Joseph G.; Allwood, Ellen G.; Hussey, Patrick J.; Hepler, Peter K.

    2006-01-01

    Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events. PMID:16920777

  8. Multi-drug Resistance Protein 4 (MRP4)-mediated Regulation of Fibroblast Cell Migration Reflects a Dichotomous Role of Intracellular Cyclic Nucleotides*

    PubMed Central

    Sinha, Chandrima; Ren, Aixia; Arora, Kavisha; Moon, Chang-Suk; Yarlagadda, Sunitha; Zhang, Weiqiang; Cheepala, Satish B.; Schuetz, John D.; Naren, Anjaparavanda P.

    2013-01-01

    It has long been known that cyclic nucleotides and cyclic nucleotide-dependent signaling molecules control cell migration. However, the concept that it is not just the absence or presence of cyclic nucleotides, but a highly coordinated balance between these molecules that regulates cell migration, is new and revolutionary. In this study, we used multidrug resistance protein 4 (MRP4)-expressing cell lines and MRP4 knock-out mice as model systems and wound healing assays as the experimental system to explore this unique and emerging concept. MRP4, a member of a large family of ATP binding cassette transporter proteins, localizes to the plasma membrane and functions as a nucleotide efflux transporter and thus plays a role in the regulation of intracellular cyclic nucleotide levels. Here, we demonstrate that mouse embryonic fibroblasts (MEFs) isolated from Mrp4−/− mice have higher intracellular cyclic nucleotide levels and migrate faster compared with MEFs from Mrp4+/+ mice. Using FRET-based cAMP and cGMP sensors, we show that inhibition of MRP4 with MK571 increases both cAMP and cGMP levels, which results in increased migration. In contrast to these moderate increases in cAMP and cGMP levels seen in the absence of MRP4, a robust increase in cAMP levels was observed following treatment with forskolin and isobutylmethylxanthine, which decreases fibroblast migration. In response to externally added cell-permeant cyclic nucleotides (cpt-cAMP and cpt-cGMP), MEF migration appears to be biphasic. Altogether, our studies provide the first experimental evidence supporting the novel concept that balance between cyclic nucleotides is critical for cell migration. PMID:23264633

  9. Cloning, nucleotide sequence, and regulation of Schizosaccharomyces pombe thi4, a thiamine biosynthetic gene.

    PubMed

    Zurlinden, A; Schweingruber, M E

    1994-11-01

    thi4 mutants of Schizosaccharomyces pombe exhibit defective thiamine biosynthesis, and thi4 mutations define a gene which is believed to be involved in the phosphorylation of 4-amino-5-hydroxymethyl-2-methylpyrimidine or 5-(2-hydroxyethyl)-4-methylthiazole and/or in the coupling of the two phosphorylated precursors to thiamine monophosphate (A. M. Schweingruber, J. Dlugonski, E. Edenharter, and M. E. Schweingruber, Curr. Genet. 19:249-254, 1991). The thi4 gene was cloned by functional complementation of a thi4 mutant and physically mapped on the left arm of chromosome I close to the genetic marker gln1. The thi4-carrying DNA fragment shows an open reading frame encoding a protein of 518 amino acids and a calculated molecular mass of 55.6 kDa. The appearance of thi4 mRNA is strongly repressed by thiamine and to a lesser extent by 5-(2-hydroxyethyl)-4-methylthiazole. thi4 mRNA production is under the control of the thi1 gene-encoded transcription factor and of the negative regulators encoded by genes tnr1, tnr2, and tnr3. thi4 is expressed and regulated in manners similar to those of other S. pombe genes involved in thiamine metabolism, including thi2, thi3, and pho4.

  10. Nucleotide sequence of the FNR-regulated fumarase gene (fumB) of Escherichia coli K-12.

    PubMed Central

    Bell, P J; Andrews, S C; Sivak, M N; Guest, J R

    1989-01-01

    The nucleotide sequence of a 3,162-base-pair (bp) segment of DNA containing the FNR-regulated fumB gene, which encodes the anaerobic class I fumarase (FUMB) of Escherichia coli, was determined. The structural gene was found to comprise 1,641 bp, 547 codons (excluding the initiation and termination codons), and the gene product had a predicted Mr of 59,956. The amino acid sequence of FUMB contained the same number of residues as did that of the aerobic class I fumarase (FUMA), and there were identical amino acids at all but 56 positions (89.8% identity). There was no significant similarity between the class I fumarases and the class II enzyme (FUMC) except in one region containing the following consensus: Gly-Ser-Xxx-Ile-Met-Xxx-Xxx-Lys-Xxx-Asn. Some of the 56 amino acid substitutions must be responsible for the functional preferences of the enzymes for malate dehydration (FUMB) and fumarate hydration (FUMA). Significant similarities between the cysteine-containing sequence of the class I fumarases (FUMA and FUMB) and the mammalian aconitases were detected, and this finding further supports the view that these enzymes are all members of a family of iron-containing hydrolyases. The nucleotide sequence of a 1,142-bp distal sequence of an unidentified gene (genF) located upstream of fumB was also defined and found to encode a product that is homologous to the product of another unidentified gene (genA), located downstream of the neighboring aspartase gene (aspA). PMID:2656658

  11. Regulation of proteasome activity by P2Y2 receptor underlies the neuroprotective effects of extracellular nucleotides.

    PubMed

    de Diego-García, Laura; Ramírez-Escudero, Mercedes; Sebastián-Serrano, Álvaro; Diaz-Hernández, Juan Ignacio; Pintor, Jesús; Lucas, José J; Díaz-Hernández, Miguel

    2017-01-01

    The Ubiquitin-Proteasome System (UPS) is essential for the regulation of the cellular proteostasis. Indeed, it has been postulated that an UPS dysregulation is the common mechanism that underlies several neurological disorders. Considering that extracellular nucleotides, through their selective P2Y2 receptor (P2Y2R), play a neuroprotective role in various neurological disorders that course with an UPS impairment, we wonder if this neuroprotective capacity resulted from their ability to modulate the UPS. Using a cellular model expressing two different UPS reporters, we found that the stimulation of P2Y2R by its selective agonist Up4U induced a significant reduction of UPS reporter levels. This reduction was due to an increase in two of the three peptidase proteasome activities, chymotrypsin and postglutamyl, caused by an increased expression of proteasome constitutive catalytic subunits β1 and β5. The intracellular signaling pathway involved required the activation of IP3/MEK1/2/ERK but was independent of PKC or PKA. Interestingly, the P2Y2R activation was able to revert both UPS-reporter accumulation and the cell death induced by a prolonged inhibition of UPS. Finally, we also observed that intracerebroventricular administration of Up4U induced a significant increase both of chymotrypsin and postglutamyl activities as well as an increased expression of proteasome subunits β1 and β5 in the hippocampus of wild-type mice, but not in P2Y2R KO mice. All these results strongly suggest that the capacity to modulate the UPS activity via P2Y2R is the molecular mechanism which is how the nucleotides play a neuroprotective role in neurological disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. C Terminus of Nucleotide Binding Domain 1 Contains Critical Features for Cystic Fibrosis Transmembrane Conductance Regulator Trafficking and Activation*

    PubMed Central

    Billet, Arnaud; Melin, Patricia; Jollivet, Mathilde; Mornon, Jean-Paul; Callebaut, Isabelle; Becq, Frédéric

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl− channel physiologically important in fluid-transporting epithelia and pathologically relevant in several human diseases. Here, we show that mutations in the C terminus of the first nucleotide binding domain comprising the latest β strands (βc5 and βc6) influence the trafficking, channel activity, and pharmacology of CFTR. We mutated CFTR amino acids located in the βc5-βc6 hairpin, within the βc5 strand (H620Q), within the β-turn linking the two β strands (E621G, G622D), as well as within (S623A, S624A) and at the extremity (G628R) of the βc6 strand. Functional analysis reveals that the current density was largely reduced for G622D and G628R channels compared with wt CFTR, similar for E621G and S624A, but increased for H620Q and S623A. For G622D and G628R, the abnormal activity is likely due to a defective maturation process, as assessed by the augmented activity and mature C-band observed in the presence of the trafficking corrector miglustat. In addition, in presence of the CFTR activator benzo[c]quinolizinium, the CFTR current density compared with that of wt CFTR was abolished for G622D and G628R channels, but similar for H620Q, S623A, and S624A or slightly increased for E621G. Finally, G622D and G628R were activated by the CFTR agonists genistein, RP-107, and isobutylmethylxanthine. Our results identify the C terminus of the CFTR first nucleotide binding domain as an important molecular site for the trafficking of CFTR protein, for the control of CFTR channel gating, and for the pharmacological effect of a dual activity agent. PMID:20435887

  13. Cis-acting single nucleotide polymorphisms alter MicroRNA-mediated regulation of human brain-expressed transcripts

    PubMed Central

    Ramachandran, Shyam; Coffin, Stephanie L.; Tang, Tin-Yun; Jobaliya, Chintan D.; Spengler, Ryan M.

    2016-01-01

    Abstract Substantial variability exists in the presentation of complex neurological disorders, and the study of single nucleotide polymorphisms (SNPs) has shed light on disease mechanisms and pathophysiological variability in some cases. However, the vast majority of disease-linked SNPs have unidentified pathophysiological relevance. Here, we tested the hypothesis that SNPs within the miRNA recognition element (MRE; the region of the target transcript to which the miRNA binds) can impart changes in the expression of those genes, either by enhancing or reducing transcript and protein levels. To test this, we cross-referenced 7,153 miRNA-MRE brain interactions with the SNP database (dbSNP) to identify candidates, and functionally assessed 24 SNPs located in the 3’UTR or the coding sequence (CDS) of targets. For over half of the candidates tested, SNPs either enhanced (4 genes) or disrupted (10 genes) miRNA binding and target regulation. Additionally, SNPs causing a shift from a common to rare codon within the CDS facilitated miRNA binding downstream of the SNP, dramatically repressing target gene expression. The biological activity of the SNPs on miRNA regulation was also confirmed in induced pluripotent stem cell (iPSC) lines. These studies strongly support the notion that SNPs in the 3’UTR or the coding sequence of disease-relevant genes may be important in disease pathogenesis and should be reconsidered as candidate modifiers. PMID:28171541

  14. RIC8 is a guanine-nucleotide exchange factor for Galpha subunits that regulates growth and development in Neurospora crassa.

    PubMed

    Wright, Sara J; Inchausti, Regina; Eaton, Carla J; Krystofova, Svetlana; Borkovich, Katherine A

    2011-09-01

    Heterotrimeric (αβγ) G proteins are crucial components of eukaryotic signal transduction pathways. G-protein-coupled receptors (GPCRs) act as guanine nucleotide exchange factors (GEFs) for Gα subunits. Recently, facilitated GDP/GTP exchange by non-GPCR GEFs, such as RIC8, has emerged as an important mechanism for Gα regulation in animals. RIC8 is present in animals and filamentous fungi, such as the model eukaryote Neurospora crassa, but is absent from the genomes of baker's yeast and plants. In Neurospora, deletion of ric8 leads to profound defects in growth and asexual and sexual development, similar to those observed for a mutant lacking the Gα genes gna-1 and gna-3. In addition, constitutively activated alleles of gna-1 and gna-3 rescue many defects of Δric8 mutants. Similar to reports in Drosophila, Neurospora Δric8 strains have greatly reduced levels of G-protein subunits. Effects on cAMP signaling are suggested by low levels of adenylyl cyclase protein in Δric8 mutants and suppression of Δric8 by a mutation in the protein kinase A regulatory subunit. RIC8 acts as a GEF for GNA-1 and GNA-3 in vitro, with the strongest effect on GNA-3. Our results support a role for RIC8 in regulating GNA-1 and GNA-3 in Neurospora.

  15. Cyclic nucleotide regulation of inositol lipid metabolism in rat cerebral cortex

    SciTech Connect

    Nicchitta, C.V.; Williamson, J.R.

    1986-05-01

    The authors have investigated the effects of compounds known to elevate cAMP levels and/or activate the cAMP dependent protein kinase on resting and stimulated inositol lipid metabolism in rat cortical slices and synaptosomes. In (/sup 3/H)-myo-inositol-labeled brain slices, carbamylcholine-stimulated accumulation of (/sup 3/H)-myo-inositol 1-phosphate (IP/sub 1/) was decreased 50% by forskolin (150..mu..M) isobutylmethylxantihine (IBMX) (1mM), 8-bromo cAMP (5mM) or 8-(4-chlorophenylthio) cAMP (5mM). In studies with synaptosomes, the incorporation of (/sup 32/Pi) into phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP/sub 2/) and phosphatidic acid (PA) was also reduced in the presence of forskolin or the cAMP phosphodiesterase inhibitors IBMX and RO20-1724 (+/-)-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone. In the presence of maximally effective concentrations of these agents, steady state PIP levels were reduced by 50% and PIP/sub 2/ and PA levels by 40%. The effects of these compounds on (/sup 32/Pi) labeling of the phosphoinositides and PA are consistent with an inhibition of flux through the inositide cycle, presumably through the action of cAMP. Such an inhibition may account for the reduction in carbamylcholine sensitive IP/sub 1/ accumulation observed in brain slices. These studies indicate that in rat brain, ..beta..-adrenergic stimulation may be involved in modulating the activity of muscarinic cholinergic agonists. This regulation appears to be at the level of inositol lipid metabolism.

  16. Bacterial Rotary Export ATPases Are Allosterically Regulated by the Nucleotide Second Messenger Cyclic-di-GMP*

    PubMed Central

    Trampari, Eleftheria; Stevenson, Clare E. M.; Little, Richard H.; Wilhelm, Thomas; Lawson, David M.; Malone, Jacob G.

    2015-01-01

    The widespread second messenger molecule cyclic di-GMP (cdG) regulates the transition from motile and virulent lifestyles to sessile, biofilm-forming ones in a wide range of bacteria. Many pathogenic and commensal bacterial-host interactions are known to be controlled by cdG signaling. Although the biochemistry of cyclic dinucleotide metabolism is well understood, much remains to be discovered about the downstream signaling pathways that induce bacterial responses upon cdG binding. As part of our ongoing research into the role of cdG signaling in plant-associated Pseudomonas species, we carried out an affinity capture screen for cdG binding proteins in the model organism Pseudomonas fluorescens SBW25. The flagella export AAA+ ATPase FliI was identified as a result of this screen and subsequently shown to bind specifically to the cdG molecule, with a KD in the low micromolar range. The interaction between FliI and cdG appears to be very widespread. In addition to FliI homologs from diverse bacterial species, high affinity binding was also observed for the type III secretion system homolog HrcN and the type VI ATPase ClpB2. The addition of cdG was shown to inhibit FliI and HrcN ATPase activity in vitro. Finally, a combination of site-specific mutagenesis, mass spectrometry, and in silico analysis was used to predict that cdG binds to FliI in a pocket of highly conserved residues at the interface between two FliI subunits. Our results suggest a novel, fundamental role for cdG in controlling the function of multiple important bacterial export pathways, through direct allosteric control of export ATPase proteins. PMID:26265469

  17. Expression and regulation of epithelial Na+ channels by nucleotides in pleural mesothelial cells.

    PubMed

    Nie, Hong-Guang; Tucker, Torry; Su, Xue-Feng; Na, Tao; Peng, Ji-Bin; Smith, Peter R; Idell, Steven; Ji, Hong-Long

    2009-05-01

    Pleural effusions are commonly clinical disorders, resulting from the imbalance between pleural fluid turnover and reabsorption. The mechanisms underlying pleural fluid clearance across the mesothelium remain to be elucidated. We hypothesized that epithelial Na(+) channel (ENaC) is expressed and forms the molecular basis of the amiloride-sensitive resistance in human mesothelial cells. Our RT-PCR results showed that three ENaC subunits, namely, alpha, beta, gamma, and two delta ENaC subunits, are expressed in human primary pleural mesothelial cells, a human mesothelioma cell line (M9K), and mouse pleural tissue. In addition, Western blotting and immunofluorescence microscopy studies revealed that alpha, beta, gamma, and delta ENaC subunits are expressed in primary human mesothelial cells and M9K cells at the protein level. An amiloride-inhibitable short-circuit current was detected in M9K monolayers and mouse pleural tissues when mounted in Ussing chambers. Whole-cell patch clamp recordings showed an ENaC-like channel with an amiloride concentration producing 50% inhibition of 12 microM in M9K cells. This cation channel has a high affinity for extracellular Na+ ions (K(m): 53 mM). The ion selectivity of this channel to cations follows the same order as ENaC: Li+ > Na+ > K+. The unitary Li(+) conductance was 15 pS in on-cell patches. Four ENaC subunits form a functional Na+ channel when coinjected into Xenopus oocytes. Furthermore, we found that both forskolin and cGMP increased the short-circuit currents in mouse pleural tissues. Taken together, our data demonstrate that the ENaC channels are biochemically and functionally expressed in human pleural mesothelial cells, and can be up-regulated by cyclic AMP and cyclic GMP.

  18. Natriuretic peptides modify Pseudomonas fluorescens cytotoxicity by regulating cyclic nucleotides and modifying LPS structure

    PubMed Central

    Veron, Wilfried; Orange, Nicole; Feuilloley, Marc GJ; Lesouhaitier, Olivier

    2008-01-01

    Background Nervous tissues express various communication molecules including natriuretic peptides, i.e. Brain Natriuretic Peptide (BNP) and C-type Natriuretic Peptide (CNP). These molecules share structural similarities with cyclic antibacterial peptides. CNP and to a lesser extent BNP can modify the cytotoxicity of the opportunistic pathogen Pseudomonas aeruginosa. The psychrotrophic environmental species Pseudomonas fluorescens also binds to and kills neurons and glial cells, cell types that both produce natriuretic peptides. In the present study, we investigated the sensitivity of Pseudomonas fluorescens to natriuretic peptides and evaluated the distribution and variability of putative natriuretic peptide-dependent sensor systems in the Pseudomonas genus. Results Neither BNP nor CNP modified P. fluorescens MF37 growth or cultivability. However, pre-treatment of P. fluorescens MF37 with BNP or CNP provoked a decrease of the apoptotic effect of the bacterium on glial cells and an increase of its necrotic activity. By homology with eukaryotes, where natriuretic peptides act through receptors coupled to cyclases, we observed that cell-permeable stable analogues of cyclic AMP (dbcAMP) and cyclic GMP (8BcGMP) mimicked the effect of BNP and CNP on bacteria. Intra-bacterial concentrations of cAMP and cGMP were measured to study the involvement of bacterial cyclases in the regulation of P. fluorescens cytotoxicity by BNP or CNP. BNP provoked an increase (+49%) of the cAMP concentration in P. fluorescens, and CNP increased the intra-bacterial concentrations of cGMP (+136%). The effect of BNP and CNP on the virulence of P. fluorescens was independent of the potential of the bacteria to bind to glial cells. Conversely, LPS extracted from MF37 pre-treated with dbcAMP showed a higher necrotic activity than the LPS from untreated or 8BcGMP-pre-treated bacteria. Capillary electrophoresis analysis suggests that these different effects of the LPS may be due, at least in part, to

  19. Arabidopsis DAYU/ABERRANT PEROXISOME MORPHOLOGY9 Is a Key Regulator of Peroxisome Biogenesis and Plays Critical Roles during Pollen Maturation and Germination in Planta[W

    PubMed Central

    Li, Xin-Ran; Li, Hong-Ju; Yuan, Li; Liu, Man; Shi, Dong-Qiao; Liu, Jie; Yang, Wei-Cai

    2014-01-01

    Pollen undergo a maturation process to sustain pollen viability and prepare them for germination. Molecular mechanisms controlling these processes remain largely unknown. Here, we report an Arabidopsis thaliana mutant, dayu (dau), which impairs pollen maturation and in vivo germination. Molecular analysis indicated that DAU encodes the peroxisomal membrane protein ABERRANT PEROXISOME MORPHOLOGY9 (APEM9). DAU is transiently expressed from bicellular pollen to mature pollen during male gametogenesis. DAU interacts with peroxisomal membrane proteins PEROXIN13 (PEX13) and PEX16 in planta. Consistently, both peroxisome biogenesis and peroxisome protein import are impaired in dau pollen. In addition, the jasmonic acid (JA) level is significantly decreased in dau pollen, and the dau mutant phenotype is partially rescued by exogenous application of JA, indicating that the male sterility is mainly due to JA deficiency. In addition, the phenotypic survey of peroxin mutants indicates that the PEXs most likely play different roles in pollen germination. Taken together, these data indicate that DAU/APEM9 plays critical roles in peroxisome biogenesis and function, which is essential for JA production and pollen maturation and germination. PMID:24510720

  20. Emerging role of Cdc42-specific guanine nucleotide exchange factors as regulators of membrane trafficking in health and disease.

    PubMed

    Egorov, M V; Polishchuk, R S

    2017-04-01

    It is widely accepted that the Golgi complex operates as a main sorting station in the biosynthetic pathway. On the other hand, the Golgi complex harbors numerous signaling molecules that generate the platform for the coordination of the transduction of specific signals and of membrane transport events. A part of these processes, which require the complex integration of transport-, cytoskeleton- and polarity-associated mechanisms, is tightly regulated by molecular machineries comprising guanine nucleotide exchange factors (GEF) and their down-stream effectors, such as the small GTPase Cdc42. Dysfunction of several Cdc42-specific GEFs has been shown to cause a number of human diseases, which are associated with impaired intracellular trafficking at the level of the Golgi complex as well as in other compartments. Here we briefly overview how mutations in Cdc42-specific GEFs have an impact on the organization of intracellular trafficking fluxes and how such trafficking aberrations could be associated with a number of human disorders. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Transcription profiling of guanine nucleotide binding proteins during developmental regulation, and pesticide response in Solenopsis invicta (Hymenoptera: Formicidae)

    USDA-ARS?s Scientific Manuscript database

    Guanine nucleotide binding proteins (GNBP or G-protein) are glycoproteins anchored on the cytoplasmic cell membrane, and are mediators for many cellular processes. Complete cDNA of guanine nucleotide-binding protein gene ß-subunit (SiGNBP) was cloned and sequenced from S. invicta workers. To detect ...

  2. The nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the endoplasmic reticulum, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

    PubMed

    Reyes, Francisca; León, Gabriel; Donoso, Maribel; Brandizzí, Federica; Weber, Andreas P M; Orellana, Ariel

    2010-02-01

    Uridine 5'-diphosphate (UDP)-glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP-glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP-glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP-glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.

  3. The gall bladder cholecystokinin receptor exists in two guanine nucleotide-binding protein-regulated affinity states

    SciTech Connect

    Molero, X.; Miller, L.J. )

    1991-02-01

    To study proximal events in cholecystokinin (CCK) action on bovine gall bladder smooth muscle, we used the hormone analogue D-Tyr-Gly-((N1e28,31)CCK-26-32)-phenethyl ester (OPE), which has unique biological properties. This fully efficacious agonist differs from native CCK by not expressing supramaximal inhibition of cell shortening, yet it clearly interacts with the same receptor molecule. This was demonstrated in binding and affinity labeling studies, where both peptides label the same Mr 70,000-85,000 protein and both fully compete for binding of the other ligand. Further, its relatively high affinity for the low affinity CCK receptor permits the clear demonstration of two affinity states of a CCK receptor on a membrane preparation and makes possible evaluation of the molecular basis of these affinity states and their regulation. Analysis of homologous and heterologous binding curves performed with both CCK and OPE peptides and radioligands demonstrated the presence of two affinity states, with CCK being able to distinguish them (Kd1 = 0.48 +/- 0.04 nM and Kd2 = 56.5 +/- 7.4 nM) and OPE recognizing them equally (Kd1 = 0.94 +/- 0.31 nM and Kd2 = 0.96 +/- 0.23 nM). In the presence of nonhydrolyzable GTP analogues, there was a shift in distribution of receptors toward the low affinity state, with the total number of receptors and their absolute affinities for each peptide remaining constant. Thus, the gall bladder CCK receptor is a single molecule capable of assuming two interconvertible affinity states, regulated by a guanine nucleotide-binding protein. Two full agonists are capable of interacting with this molecule to yield different biological responses via different molecular events.

  4. Allergenic pollen and pollen allergy in Europe.

    PubMed

    D'Amato, G; Cecchi, L; Bonini, S; Nunes, C; Annesi-Maesano, I; Behrendt, H; Liccardi, G; Popov, T; van Cauwenberge, P

    2007-09-01

    The allergenic content of the atmosphere varies according to climate, geography and vegetation. Data on the presence and prevalence of allergenic airborne pollens, obtained from both aerobiological studies and allergological investigations, make it possible to design pollen calendars with the approximate flowering period of the plants in the sampling area. In this way, even though pollen production and dispersal from year to year depend on the patterns of preseason weather and on the conditions prevailing at the time of anthesis, it is usually possible to forecast the chances of encountering high atmospheric allergenic pollen concentrations in different areas. Aerobiological and allergological studies show that the pollen map of Europe is changing also as a result of cultural factors (for example, importation of plants such as birch and cypress for urban parklands), greater international travel (e.g. colonization by ragweed in France, northern Italy, Austria, Hungary etc.) and climate change. In this regard, the higher frequency of weather extremes, like thunderstorms, and increasing episodes of long range transport of allergenic pollen represent new challenges for researchers. Furthermore, in the last few years, experimental data on pollen and subpollen-particles structure, the pathogenetic role of pollen and the interaction between pollen and air pollutants, gave new insights into the mechanisms of respiratory allergic diseases.

  5. Allergies, asthma, and pollen

    MedlinePlus

    ... hay fever (allergic rhinitis) and asthma include: Some trees Some grasses Weeds Ragweed ... pollen at different times of the year. Most trees produce pollen in the spring. Grasses usually produce ...

  6. The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

    PubMed Central

    Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

    2014-01-01

    Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

  7. Mitotic regulator Nlp interacts with XPA/ERCC1 complexes and regulates nucleotide excision repair (NER) in response to UV radiation.

    PubMed

    Ma, Xiao-Juan; Shang, Li; Zhang, Wei-Min; Wang, Ming-Rong; Zhan, Qi-Min

    2016-04-10

    Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER.

  8. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro *

    PubMed Central

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-01-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved. PMID:26792808

  9. Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro.

    PubMed

    Fíla, Jan; Radau, Sonja; Matros, Andrea; Hartmann, Anja; Scholz, Uwe; Feciková, Jana; Mock, Hans-Peter; Čapková, Věra; Zahedi, René Peiman; Honys, David

    2016-04-01

    Tobacco mature pollen has extremely desiccated cytoplasm, and is metabolically quiescent. Upon re-hydration it becomes metabolically active and that results in later emergence of rapidly growing pollen tube. These changes in cytoplasm hydration and metabolic activity are accompanied by protein phosphorylation. In this study, we subjected mature pollen, 5-min-activated pollen, and 30-min-activated pollen to TCA/acetone protein extraction, trypsin digestion and phosphopeptide enrichment by titanium dioxide. The enriched fraction was subjected to nLC-MS/MS. We identified 471 phosphopeptides that carried 432 phosphorylation sites, position of which was exactly matched by mass spectrometry. These 471 phosphopeptides were assigned to 301 phosphoproteins, because some proteins carried more phosphorylation sites. Of the 13 functional groups, the majority of proteins were put into these categories: transcription, protein synthesis, protein destination and storage, and signal transduction. Many proteins were of unknown function, reflecting the fact that male gametophyte contains many specific proteins that have not been fully functionally annotated. The quantitative data highlighted the dynamics of protein phosphorylation during pollen activation; the identified phosphopeptides were divided into seven groups based on the regulatory trends. The major group comprised mature pollen-specific phosphopeptides that were dephosphorylated during pollen activation. Several phosphopeptides representing the same phosphoprotein had different regulation, which pinpointed the complexity of protein phosphorylation and its clear functional context. Collectively, we showed the first phosphoproteomics data on activated pollen where the position of phosphorylation sites was clearly demonstrated and regulatory kinetics was resolved.

  10. Pollen lipidomics: lipid profiling exposes a notable diversity in 22 allergenic pollen and potential biomarkers of the allergic immune response.

    PubMed

    Bashir, Mohamed Elfatih H; Lui, Jan Hsi; Palnivelu, Ravishankar; Naclerio, Robert M; Preuss, Daphne

    2013-01-01

    Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins. We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture. Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen lipids vary greatly among allergenic species and contain many molecules

  11. Pollen Lipidomics: Lipid Profiling Exposes a Notable Diversity in 22 Allergenic Pollen and Potential Biomarkers of the Allergic Immune Response

    PubMed Central

    Bashir, Mohamed Elfatih H.; Lui, Jan Hsi; Palnivelu, Ravishankar; Naclerio, Robert M.; Preuss, Daphne

    2013-01-01

    Background/Aim Pollen grains are the male gametophytes that deliver sperm cells to female gametophytes during sexual reproduction of higher plants. Pollen is a major source of aeroallergens and environmental antigens. The pollen coat harbors a plethora of lipids that are required for pollen hydration, germination, and penetration of the stigma by pollen tubes. In addition to proteins, pollen displays a wide array of lipids that interact with the human immune system. Prior searches for pollen allergens have focused on the identification of intracellular allergenic proteins, but have largely overlooked much of the extracellular pollen matrix, a region where the majority of lipid molecules reside. Lipid antigens have attracted attention for their potent immunoregulatory effects. By being in close proximity to allergenic proteins on the pollen surface when they interact with host cells, lipids could modify the antigenic properties of proteins. Methodology/Principal Findings We performed a comparative pollen lipid profiling of 22 commonly allergenic plant species by the use of gas chromatography-mass spectroscopy, followed by detailed data mining and statistical analysis. Three experiments compared pollen lipid profiles. We built a database library of the pollen lipids by matching acquired pollen-lipid mass spectra and retention times with the NIST/EPA/NIH mass-spectral library. We detected, identified, and relatively quantified more than 106 lipid molecular species including fatty acids, n-alkanes, fatty alcohols, and sterols. Pollen-derived lipids stimulation up-regulate cytokines expression of dendritic and natural killer T cells co-culture. Conclusions/Significance Here we report on a lipidomic analysis of pollen lipids that can serve as a database for identifying potential lipid antigens and/or novel candidate molecules involved in allergy. The database provides a resource that facilitates studies on the role of lipids in the immunopathogenesis of allergy. Pollen

  12. Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells.

    PubMed

    Sanchez-Aguilera, Abel; Lee, Yun-Jung; Lo Celso, Cristina; Ferraro, Francesca; Brumme, Kristina; Mondal, Subhanjan; Kim, Chaekyun; Dorrance, Adrienne; Luo, Hongbo R; Scadden, David T; Williams, David A

    2011-06-07

    Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1(-/-) HSPC showed impaired early localization near nestin(+) perivascular mesenchymal stem cells; only 6.25% of Vav1(-/-) HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin(+) cell. Abnormal perivascular localization correlated with decreased retention of Vav1(-/-) HSPC in the bone marrow (44-60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1(-/-) cells vs. 53-63% for wild-type cells). The engraftment defect of Vav1(-/-) HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1(-/-) HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment.

  13. Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells

    PubMed Central

    Sanchez-Aguilera, Abel; Lee, Yun-Jung; Lo Celso, Cristina; Ferraro, Francesca; Brumme, Kristina; Mondal, Subhanjan; Kim, Chaekyun; Dorrance, Adrienne; Luo, Hongbo R.; Scadden, David T.; Williams, David A.

    2011-01-01

    Engraftment and maintenance of hematopoietic stem and progenitor cells (HSPC) depend on their ability to respond to extracellular signals from the bone marrow microenvironment, but the critical intracellular pathways integrating these signals remain poorly understood. Furthermore, recent studies provide contradictory evidence of the roles of vascular versus osteoblastic niche components in HSPC function. To address these questions and to dissect the complex upstream regulation of Rac GTPase activity in HSPC, we investigated the role of the hematopoietic-specific guanine nucleotide exchange factor Vav1 in HSPC localization and engraftment. Using intravital microscopy assays, we demonstrated that transplanted Vav1−/− HSPC showed impaired early localization near nestin+ perivascular mesenchymal stem cells; only 6.25% of Vav1−/− HSPC versus 45.8% of wild-type HSPC were located less than 30 μm from a nestin+ cell. Abnormal perivascular localization correlated with decreased retention of Vav1−/− HSPC in the bone marrow (44–60% reduction at 48 h posttransplant, compared with wild-type) and a very significant defect in short- and long-term engraftment in competitive and noncompetitive repopulation assays (<1.5% chimerism of Vav1−/− cells vs. 53–63% for wild-type cells). The engraftment defect of Vav1−/− HSPC was not related to alterations in proliferation, survival, or integrin-mediated adhesion. However, Vav1−/− HSPC showed impaired responses to SDF1α, including reduced in vitro migration in time-lapse microscopy assays, decreased circadian and pharmacologically induced mobilization in vivo, and dysregulated Rac/Cdc42 activation. These data suggest that Vav1 activity is required specifically for SDF1α-dependent perivascular homing of HSPC and suggest a critical role for this localization in retention and subsequent engraftment. PMID:21606370

  14. Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1995-01-01

    1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

  15. Regulation of cyclic nucleotide-gated channels and membrane excitability in olfactory receptor cells by carbon monoxide

    NASA Technical Reports Server (NTRS)

    Leinders-Zufall, T.; Shepherd, G. M.; Zufall, F.

    1995-01-01

    1. The effect of the putative neural messenger carbon monoxide (CO) and the role of the cGMP second-messenger system for olfactory signal generation was examined in isolated olfactory receptor neurons (ORNs) of the tiger salamander. 2. With the use of whole cell voltage-clamp recordings in combination with a series of ionic and pharmological tests, it is demonstrated that exogenously applied CO is a potent activator (K1/2 = 2.9 microM) of cyclic nucleotide-gated (CNG) channels previously described to mediate odor transduction. 3. Several lines of evidence suggest that CO mediates its effect through stimulation of a soluble guanylyl cyclase (sGC) leading to formation of the second-messenger cGMP. This conclusion is based on the findings that CO responses show an absolute requirement for guanosine 5'-triphosphate (GTP) in the internal solution, that no direct effect of CO on CNG currents in the absence of GTP is detectable, and that a blocker of sGC activation, LY85383 (10 microM), completely inhibits the CO response. 4. The dose-response curve for cGMP at CNG channels is used as a calibration to provide a quantitative estimate of the CO-stimulated cGMP formation. This analysis implies that CO is a potent activator of olfactory sGC. 5. Perforated patch recordings using amphotericin B demonstrate that low micromolar doses of CO effectively depolarize the membrane potential of ORNs through tonic activation of CNG channels. This effect in turn regulates excitable and adaptive properties of ORNs and modulates neuronal responsiveness. 6. These data argue for an important role of the cGMP pathway in olfactory signaling and support the idea that CO may function as a diffusible messenger in the olfactory system.

  16. Plant Cyclic Nucleotide Signalling

    PubMed Central

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc

    2007-01-01

    The presence of the cyclic nucleotides 3′,5′-cyclic adenyl monophosphate (cAMP) and 3′,5′-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed. PMID:19704553

  17. Pollen tube development.

    PubMed

    Johnson, Mark A; Kost, Benedikt

    2010-01-01

    Pollen tubes grow rapidly in a strictly polarized manner as they transport male reproductive cells through female flower tissues to bring about fertilization. Vegetative pollen tube cells are an excellent model system to investigate processes underlying directional cell expansion. In this chapter, we describe materials and methods required for (1) the identification of novel factors essential for polarized cell growth through the isolation and analysis of Arabidopsis mutants with defects in pollen tube growth and (2) the detailed functional characterization of pollen tube proteins based on transient transformation and microscopic analysis of cultured tobacco pollen tubes.

  18. Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1.

    PubMed

    Fleming, Ian N; Batty, Ian H; Prescott, Alan R; Gray, Alex; Kular, Gursant S; Stewart, Hazel; Downes, C Peter

    2004-09-15

    Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P(2) to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P(2) breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P(3) concentrations, rather than the closely related lipid, PtdIns(3,4)P(2). Finally, the data demonstrate that PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3) bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo.

  19. Inositol phospholipids regulate the guanine-nucleotide-exchange factor Tiam1 by facilitating its binding to the plasma membrane and regulating GDP/GTP exchange on Rac1

    PubMed Central

    2004-01-01

    Binding of the Rac1-specific guanine-nucleotide-exchange factor, Tiam1, to the plasma membrane requires the N-terminal pleckstrin homology domain. In the present study, we show that membrane-association is mediated by binding of PtdIns(4,5)P2 to the pleckstrin homology domain. Moreover, in 1321N1 astrocytoma cells, translocation of Tiam1 to the cytosol, following receptor-mediated stimulation of PtdIns(4,5)P2 breakdown, correlates with decreased Rac1-GTP levels, indicating that membrane-association is required for GDP/GTP exchange on Rac1. In addition, we show that platelet-derived growth factor activates Rac1 in vivo by increasing PtdIns(3,4,5)P3 concentrations, rather than the closely related lipid, PtdIns(3,4)P2. Finally, the data demonstrate that PtdIns(4,5)P2 and PtdIns(3,4,5)P3 bind to the same pleckstrin homology domain in Tiam1 and that soluble inositol phosphates appear to compete with lipids for this binding. Together, these novel observations provide strong evidence that distinct phosphoinositides regulate different functions of this enzyme, indicating that local concentrations of signalling lipids and the levels of cytosolic inositol phosphates will play crucial roles in determining its activity in vivo. PMID:15242348

  20. Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)*

    PubMed Central

    Carter, Angela M.; Gutowski, Stephen; Sternweis, Paul C.

    2014-01-01

    The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA. PMID:24855647

  1. Considerations About Pollen Used for the Production of Allergen Extracts.

    PubMed

    Codina, Rosa; Crenshaw, Rodger C; Lockey, Richard F

    2015-01-01

    Pollen is a biological product obtained to manufacture tree, weed, and grass allergen extracts, used to diagnose and treat allergies. Genetic and environmental factors affect the composition of pollen, e.g., the plant varieties from which pollen are obtained, weather, and levels of air pollution during plant growth. Therefore, appropriate guidelines and training of personnel to perform the activities associated with pollen are essential to produce appropriate allergen extracts. Various regulatory institutions, which vary in different countries, including the Food and Drug Administration (FDA) in the USA, control how such products should be produced. For example, the FDA regulates the manufacturing of pollen extracts but not the quality of the pollen used to prepare them, relying on each manufacturer to set its own standards to do so. To the contrary, European regulatory agencies, including the European Medicines Agency, control both the quality of the pollen and the manufacturing process to produce pollen extracts. Regulatory agencies, allergen manufacturers, scientific institutions, and pollen collection entities should collaborate to develop and implement guidelines appropriate for worldwide use for both the collection and processing of pollen raw materials. This article provides an overview of the subject of pollen for use in allergen extracts.

  2. Native mass spectrometry provides direct evidence for DNA mismatch-induced regulation of asymmetric nucleotide binding in mismatch repair protein MutS.

    PubMed

    Monti, Maria Chiara; Cohen, Serge X; Fish, Alexander; Winterwerp, Herrie H K; Barendregt, Arjan; Friedhoff, Peter; Perrakis, Anastassis; Heck, Albert J R; Sixma, Titia K; van den Heuvel, Robert H H; Lebbink, Joyce H G

    2011-10-01

    The DNA mismatch repair protein MutS recognizes mispaired bases in DNA and initiates repair in an ATP-dependent manner. Understanding of the allosteric coupling between DNA mismatch recognition and two asymmetric nucleotide binding sites at opposing sides of the MutS dimer requires identification of the relevant MutS.mmDNA.nucleotide species. Here, we use native mass spectrometry to detect simultaneous DNA mismatch binding and asymmetric nucleotide binding to Escherichia coli MutS. To resolve the small differences between macromolecular species bound to different nucleotides, we developed a likelihood based algorithm capable to deconvolute the observed spectra into individual peaks. The obtained mass resolution resolves simultaneous binding of ADP and AMP.PNP to this ABC ATPase in the absence of DNA. Mismatched DNA regulates the asymmetry in the ATPase sites; we observe a stable DNA-bound state containing a single AMP.PNP cofactor. This is the first direct evidence for such a postulated mismatch repair intermediate, and showcases the potential of native MS analysis in detecting mechanistically relevant reaction intermediates.

  3. Sphingomyelin Phosphodiesterase Acid-like 3A (SMPDL3A) Is a Novel Nucleotide Phosphodiesterase Regulated by Cholesterol in Human Macrophages*

    PubMed Central

    Traini, Mathew; Quinn, Carmel M.; Sandoval, Cecilia; Johansson, Erik; Schroder, Kate; Kockx, Maaike; Meikle, Peter J.; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis. PMID:25288789

  4. Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a novel nucleotide phosphodiesterase regulated by cholesterol in human macrophages.

    PubMed

    Traini, Mathew; Quinn, Carmel M; Sandoval, Cecilia; Johansson, Erik; Schroder, Kate; Kockx, Maaike; Meikle, Peter J; Jessup, Wendy; Kritharides, Leonard

    2014-11-21

    Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Binding of the P2Y2 Nucleotide Receptor to Filamin A Regulates Migration of Vascular Smooth Muscle Cells

    PubMed Central

    Yu, Ningpu; Erb, Laurie; Shivaji, Rikka; Weisman, Gary A.; Seye, Cheikh I.

    2013-01-01

    The functional expression of the G protein– coupled P2Y2 nucleotide receptor (P2Y2R) has been associated with proliferation and migration of vascular smooth muscle cells (SMCs), 2 processes involved in atherosclerosis and restenosis. Activation of the P2Y2R causes dynamic reorganization of the actin cytoskeleton, which transmits biochemical signals and forces necessary for cell locomotion, suggesting that P2Y2Rs may be linked to the actin cytoskeleton. Here, we identified filamin A (FLNa) as a P2Y2R-interacting protein using a yeast 2-hybrid system screen with the C-terminal region of the P2Y2R as bait. The FLNa binding site in the P2Y2R is localized between amino acids 322 and 333. Deletion of this region led to selective loss of FLNa binding to the P2Y2R and abolished Tyr phosphorylation of FLNa induced by the P2Y2R agonist UTP. Using both time-lapse microscopy and the Transwell cell migration assay, we showed that UTP significantly increased SMC spreading on collagen I (6.8 fold; P≤0.01) and migration (3.6 fold; P≤0.01) of aortic SMCs isolated from wild-type mice, as compared with unstimulated SMCs. UTP-induced spreading and migration of aortic SMCs did not occur with cells isolated from P2Y2R knockout mice. Expression of the full-length P2Y2R in SMCs isolated from P2Y2R knockout mice restored both UTP-induced spreading and migration. In contrast, UTP-induced spreading and migration did not occur in SMCs isolated from P2Y2R knockout mice transfected with a mutant P2Y2R that does not bind FLNa. Furthermore, ex vivo studies showed that both ATP and UTP (10 µmol/L) promoted migration of SMCs out of aortic explants isolated from wild-type but not P2Y2R knockout mice. Thus, this study demonstrates that P2Y2R/FLNa interaction selectively regulates spreading and migration of vascular SMCs. PMID:18202316

  6. Social memory, amnesia, and autism: brain oxytocin secretion is regulated by NAD+ metabolites and single nucleotide polymorphisms of CD38.

    PubMed

    Higashida, Haruhiro; Yokoyama, Shigeru; Huang, Jian-Jun; Liu, Li; Ma, Wen-Jie; Akther, Shirin; Higashida, Chiharu; Kikuchi, Mitsuru; Minabe, Yoshio; Munesue, Toshio

    2012-11-01

    Previously, we demonstrated that CD38, a transmembrane protein with ADP-ribosyl cyclase activity, plays a critical role in mouse social behavior by regulating the release of oxytocin (OXT), which is essential for mutual recognition. When CD38 was disrupted, social amnesia was observed in Cd38 knockout mice. The autism spectrum disorders (ASDs), characterized by defects in reciprocal social interaction and communication, occur either sporadically or in a familial pattern. However, the etiology of ASDs remains largely unknown. Therefore, the theoretical basis for pharmacological treatments has not been established. Hence, there is a rationale for investigating single nucleotide polymorphisms (SNPs) in the human CD38 gene in ASD subjects. We found several SNPs in this gene. The SNP rs3796863 (C>A) was associated with high-functioning autism (HFA) in American samples from the Autism Gene Resource Exchange. Although this finding was partially confirmed in low-functioning autism subjects in Israel, it has not been replicated in Japanese HFA subjects. The second SNP of interest, rs1800561 (4693C>T), leads to the substitution of an arginine (R) at codon 140 by tryptophan (W; R140W) in CD38. This mutation was found in four probands of ASD and in family members of three pedigrees with variable levels of ASD or ASD traits. The plasma levels of OXT in ASD subjects with the R140W allele were lower than those in ASD subjects lacking this allele. The OXT levels were unchanged in healthy subjects with or without this mutation. One proband with the R140W allele receiving intranasal OXT for approximately 3years showed improvement in areas of social approach, eye contact and communication behaviors, emotion, irritability, and aggression. Five other ASD subjects with mental deficits received nasal OXT for various periods; three subjects showed improved symptoms, while two showed little or no effect. These results suggest that SNPs in CD38 may be possible risk factors for ASD by

  7. On the interactions between nucleotide binding domains and membrane spanning domains in cystic fibrosis transmembrane regulator: A molecular dynamic study.

    PubMed

    Belmonte, Luca; Moran, Oscar

    2015-04-01

    The Cystic Fibrosis Transmembrane Regulator (CFTR) is a membrane protein whose mutations cause cystic fibrosis, a lethal genetic disease. We performed a molecular dynamic (MD) study of the properties of the nucleotide binding domains (NBD) whose conformational changes, upon ATP binding, are the direct responsible of the gating mechanisms of CFTR. This study was done for the wild type (WT) CFTR and for the two most common mutations, ΔF508, that produces a traffic defect of the protein, and the mutation G551D, that causes a gating defect on CFTR. Using an homology model of the open channel conformation of the CFTR we thus introduced the mutations to the structure. Although the overall structures of the G551D and ΔF508 are quite well conserved, the NBD1-NBD2 interactions are severely modified in both mutants. NBD1 and NBD2 are indeed destabilized with a higher internal energy (Ei) in the ΔF508-CFTR. Differently, Ei does not change in the NBDs of G551D but, while the number of close contacts between NBD1 and NBD2 in ΔF508 is increased, a significant reduction of close contacts is found in the G551D mutated form. Hydrogen bonds formation between NBDs of the two mutated forms is also altered and it is slightly increased for the ΔF508, while are severely reduced in G551D. A consequent modification of the NBDs-ICLs interactions between residues involved in the transduction of the ATP binding and the channel gating is also registered. Indeed, while a major interaction is noticed between NBDs interface and ICL2 and ICL4 in the WT, this interaction is somehow altered in both mutated forms plausibly with effect on channel gating. Thus, single point mutations of the CFTR protein can reasonably results in channel gating defects due to alteration of the interaction mechanisms between the NBDs and NBDs-ICLs interfaces upon ATP-binding process. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  8. Gene expression profile indicates involvement of NO in Camellia sinensis pollen tube growth at low temperature.

    PubMed

    Pan, Junting; Wang, Weidong; Li, Dongqin; Shu, Zaifa; Ye, Xiaoli; Chang, Pinpin; Wang, Yuhua

    2016-10-18

    Nitric oxide (NO) functions as a critical signaling molecule in the low-temperature stress responses in plants, including polarized pollen tube growth in Camellia sinensis. Despite this, the potential mechanisms underlying the participation of NO in pollen tube responses to low temperature remain unclear. Here, we investigate alterations to gene expression in C. sinensis pollen tubes exposed to low-temperature stress and NO using RNA-Seq technology, in order to find the potential candidate genes related to the regulation of pollen tube elongation by NO under low-temperature stress. Three libraries were generated from C. sinensis cv. 'Longjingchangye' pollen tubes cultured at 25 °C (CsPT-CK) and 4 °C (CsPT-LT) or with 25 μM DEA NONOate (CsPT-NO). The number of unigenes found for the three biological replications were 39,726, 40,440 and 41,626 for CsPT-CK; 36,993, 39,070 and 39,439 for CsPT-LT; and 39,514, 38,298 and 39,061 for CsPT-NO. A total of 36,097 unique assembled and annotated sequences from C. sinensis pollen tube reads were found in a BLAST search of the following databases: NCBI non-redundant nucleotide, Swiss-prot protein, Kyoto Encyclopedia of Genes and Genomes, Cluster of Orthologous Groups of proteins, and Gene Ontology. The absolute values of log2Ratio > 1 and probability > 0.7 were used as the thresholds for significantly differential gene expression, and 766, 497 and 929 differentially expressed genes (DEGs) were found from the comparison analyses of the CK-VS-LT, CK-VS-NO and LT-VS-NO libraries, respectively. Genes related to metabolism and signaling pathways of plant hormones, transcription factors (TFs), vesicle polarized trafficking, cell wall biosynthesis, the ubiquitination machinery of the ubiquitin system and species-specific secondary metabolite pathways were mainly observed in the CK-VS-LT and CK-VS-NO libraries. Differentially expressed unigenes related to the inhibition of C. sinensis pollen tube growth under low

  9. A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1

    PubMed Central

    Reeder, Sarah H.; Lee, Byung Ha; Fox, Ronald; Dobritsa, Anna A.

    2016-01-01

    Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them. PMID:27177036

  10. [Birch pollen allergy].

    PubMed

    Lavaud, F; Fore, M; Fontaine, J-F; Pérotin, J M; de Blay, F

    2014-02-01

    In the North-East of France, birch is the main tree responsible of spring pollen allergy. However, the epidemiology of sensitization to birch pollen remains unclear. Monosensitization to birch pollen seems rare because of the frequency of cross-reactions with other pollens of the same botanical family via the major allergen Bet v 1. Around one third of patients with allergic rhinoconjunctivitis due to birch pollen are also asthmatics and a half suffer from a food allergy, essentially an oral syndrome due to rosaceae fruits eaten raw. The molecular allergens of birch pollen are well-known and have been cloned. They are available for use in in vitro diagnostic tests and also in clinical trials of specific immunotherapy.

  11. Genomic expression profiling of mature soybean (Glycine max) pollen

    PubMed Central

    Haerizadeh, Farzad; Wong, Chui E; Bhalla, Prem L; Gresshoff, Peter M; Singh, Mohan B

    2009-01-01

    Background Pollen, the male partner in the reproduction of flowering plants, comprises either two or three cells at maturity. The current knowledge of the pollen transcriptome is limited to the model plant systems Arabidopsis thaliana and Oryza sativa which have tri-cellular pollen grains at maturity. Comparative studies on pollen of other genera, particularly crop plants, are needed to understand the pollen gene networks that are subject to functional and evolutionary conservation. In this study, we used the Affymetrix Soybean GeneChip® to perform transcriptional profiling on mature bi-cellular soybean pollen. Results Compared to the sporophyte transcriptome, the soybean pollen transcriptome revealed a restricted and unique repertoire of genes, with a significantly greater proportion of specifically expressed genes than is found in the sporophyte tissue. Comparative analysis shows that, among the 37,500 soybean transcripts addressed in this study, 10,299 transcripts (27.46%) are expressed in pollen. Of the pollen-expressed sequences, about 9,489 (92.13%) are also expressed in sporophytic tissues, and 810 (7.87%) are selectively expressed in pollen. Overall, the soybean pollen transcriptome shows an enrichment of transcription factors (mostly zinc finger family proteins), signal recognition receptors, transporters, heat shock-related proteins and members of the ubiquitin proteasome proteolytic pathway. Conclusion This is the first report of a soybean pollen transcriptional profile. These data extend our current knowledge regarding regulatory pathways that govern the gene regulation and development of pollen. A comparison between transcription factors up-regulated in soybean and those in Arabidopsis revealed some divergence in the numbers and kinds of regulatory proteins expressed in both species. PMID:19265555

  12. [Regional and extra-local pollen in tundra pollen samples].

    PubMed

    Vasil'chuk, A K

    2005-01-01

    Patterns of pollen spectra formation in the tundra zone of Eurasia were considered. Changes in total pollen concentration were traced in subfossil pollen samples of the tundra zone. The data on subfossil pollen spectra were used to evaluate the proportion between local and regional plus extra-local components of tundra pollen samples as well as the changes in concentration of pollen of Scots and Siberian stone pines as well as of tree and shrub birches. The diameter of dwarf birch pollen was determined in different tundra subzones of Western Siberia. The role of extra-local and regional pollen was considered for all vegetation subzones of tundra.

  13. In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts.

    PubMed

    Fan, L M; Wang, Y F; Wang, H; Wu, W H

    2001-08-01

    The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.

  14. Dating Fossil Pollen: A Simulation.

    ERIC Educational Resources Information Center

    Sheridan, Philip

    1992-01-01

    Describes a hands-on simulation in which students determine the age of "fossil" pollen samples based on the pollen types present when examined microscopically. Provides instructions for the preparation of pollen slides. (MDH)

  15. A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style.

    PubMed

    Eberle, Carrie A; Clasen, Benjamin M; Anderson, Neil O; Smith, Alan G

    2012-03-01

    Sexual plant reproduction requires multiple pollen-pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

  16. Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels.

    PubMed

    Chinopoulos, Christos; Konràd, Csaba; Kiss, Gergely; Metelkin, Eugeniy; Töröcsik, Beata; Zhang, Steven F; Starkov, Anatoly A

    2011-04-01

    Cyclophilin D was recently shown to bind to and decrease the activity of F(0)F(1)-ATP synthase in submitochondrial particles and permeabilized mitochondria [Giorgio V et al. (2009) J Biol Chem, 284, 33982-33988]. Cyclophilin D binding decreased both ATP synthesis and hydrolysis rates. In the present study, we reaffirm these findings by demonstrating that, in intact mouse liver mitochondria energized by ATP, the absence of cyclophilin D or the presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria, an increase in F(0)F(1)-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident in the form of slightly increased respiration rates during arsenolysis. However, the modulation of F(0)F(1)-ATP synthase by cyclophilin D did not increase the adenine nucleotide translocase (ANT)-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of an effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ∼ 2.2-fold lower flux control coefficient of the F(0)F(1)-ATP synthase than that of ANT, as deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that an ∼ 30% change in F(0)F(1)-ATP synthase activity in fully energized or fully de-energized mitochondria affects the ADP-ATP exchange rate mediated by the ANT in the range 1.38-1.7%. We conclude that, in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or the inhibition of its binding to F(0)F(1)-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels. © 2011 The Authors Journal compilation © 2011 FEBS.

  17. Regulation of a volume-sensitive anion channel in rat pancreatic β-cells by intracellular adenine nucleotides

    PubMed Central

    Miley, Helen E; Brown, Peter D; Best, Leonard

    1999-01-01

    The patch-clamp technique in the whole-cell configuration was used to measure the effects of intracellular adenine nucleotides on activity of the volume-sensitive anion channel in single, isolated rat pancreatic β-cells. In the absence of intracellular nucleotides, swelling of cells with a hypertonic pipette solution failed to activate the conductance. Addition of ATP over the range 2–10 mM maintaining the same degree of hypertonicity caused a progressive activation of the conductance. An increase in ATP produced a similar activation of the conductance in non-swollen cells, albeit with reduced current amplitudes. Activation of the conductance was also observed in the presence of ATPγS, adenylyl imidophosphate (AMP-PNP), ADP, diadenosine tetraphosphate and GTPγS. Neither ADP nor GDPβS inhibited activation of the conductance by ATP. It is concluded that activity of the β-cell volume-sensitive anion channel can be modulated by changes in intracellular concentrations of ATP within the physiological concentration range by a mechanism that does not require nucleotide hydrolysis. Activity of the channel does not appear to be modulated by a G protein-coupled mechanism. PMID:10050008

  18. Gametophytic Pollen Tube Guidance: Attractant Peptides, Gametic Controls, and Receptors1[OPEN

    PubMed Central

    2017-01-01

    Pollen tube guidance in flowering plants is a unique and critical process for successful sexual reproduction. The pollen tube that grows from pollen, which is the male gametophyte, precisely navigates to the embryo sac, which is the female gametophyte, within the pistil. Recent advances have clarified the molecular framework of gametophytic pollen tube guidance. Multiple species-specific attractant peptides are secreted from synergid cells, the proper development and function of which are regulated by female gametes. Multiple receptor-like kinases on the pollen tube tip are involved in sensing species-specific attractant peptides. In this Update article, recent progress in our understanding of the mechanism of gametophytic pollen tube guidance is reviewed, including attraction by synergid cells, control of pollen tube guidance by female gametes, and directional growth of the pollen tube by directional cue sensing. Future directions in the study of pollen tube guidance also are discussed. PMID:27920159

  19. The Rho guanine nucleotide exchange factors Intersectin 1L and β-Pix control calcium-regulated exocytosis in neuroendocrine PC12 cells.

    PubMed

    Momboisse, F; Ory, S; Ceridono, M; Calco, V; Vitale, N; Bader, M-F; Gasman, S

    2010-11-01

    GTPases of the Rho family are molecular switches that play an important role in a wide range of membrane-trafficking processes including neurotransmission and hormone release. We have previously demonstrated that RhoA and Cdc42 regulate calcium-dependent exocytosis in chromaffin cells by controlling actin dynamics, whereas Rac1 regulates lipid organisation. These findings raised the question of the upstream mechanism activating these GTPases during exocytosis. The guanine nucleotide exchange factors (GEFs) that catalyse the exchange of GDP for GTP are crucial elements regulating Rho signalling. Using an RNA interference approach, we have recently demonstrated that the GEFs Intersectin-1L and β-Pix, play essential roles in neuroendocrine exocytosis by controlling the activity of Cdc42 and Rac1, respectively. This review summarizes these results and discusses the functional importance of Rho GEFs in the exocytotic machinery in neuroendocrine cells.

  20. NaStEP: a proteinase inhibitor essential to self-incompatibility and a positive regulator of HT-B stability in Nicotiana alata pollen tubes.

    PubMed

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

    2013-01-01

    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown.

  1. Reduction in allergenicity of grass pollen by genetic engineering.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    2001-01-01

    Hay fever and allergic asthma triggered by grass pollen allergens affect approximately 20% of the population in cool temperate climates. Ryegrass is the dominant source of allergens due to its prodigious airborne pollen production. Lol p 5 or group 5 is among the most important and widespread grass pollen allergen because it reacts with IgE antibodies of more than 90% of grass pollen-allergic patients, contains most of the grass pollen-specific IgE epitopes and elicits strong biological responses. Significant efforts have been made in developing diagnostic and therapeutic reagents for designing new and more effective immunotherapeutic strategies for treatment of allergic diseases. An alternative approach to this problem could be to reduce the amount of allergen content in the source plant. High velocity microprojectile bombardment was used to genetically engineer ryegrass. Antisense construct targeted to one of major allergen, Lol p 5, was introduced. The expression of antisense RNA was regulated by a pollen-specific promoter. Pollen was analysed for IgE reactivity. Analysis of proteins with allergen-specific monoclonal and polyclonal antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE binding capacity of pollen extract as compared to control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development. Our studies showed that it is possible to selectively 'switch off' allergen production in pollen of ryegrass demonstrating feasibility of genetic engineering of plants for reduced allergenicity. Copyright 2001 S. Karger AG, Basel

  2. Interaction between the AAA+ ATPase p97 and its cofactor ataxin3 in health and disease: Nucleotide-induced conformational changes regulate cofactor binding.

    PubMed

    Rao, Maya V; Williams, Dewight R; Cocklin, Simon; Loll, Patrick J

    2017-09-22

    p97 is an essential ATPase associated with various cellular activities (AAA+) that functions as a segregase in diverse cellular processes, including the maintenance of proteostasis. p97 interacts with different cofactors that target it to distinct pathways; an important example is the deubiquitinase ataxin3, which collaborates with p97 in endoplasmic reticulum associated degradation (ERAD). However, the molecular details of this interaction have been unclear. Here, we characterized the binding of ataxin3 to p97, showing that ataxin3 binds with low-micromolar affinity to both wild-type p97 and mutants linked to degenerative disorders known as multisystem proteinopathy 1 (MSP1); we further showed that the stoichiometry of binding is one ataxin3 molecule per p97 hexamer. We mapped the binding determinants on each protein, demonstrating that ataxin3's p97/VCP-binding motif (VBM) interacts with the inter-lobe cleft in the N-domain of p97. We also probed the nucleotide dependence of this interaction, confirming that ataxin3 and p97 associate in the presence of ATP and in the absence of nucleotide, but not in the presence of ADP. Our experiments suggest that an ADP-driven downward movement of the p97 N-terminal domain dislodges ataxin3 by inducing a steric clash between the D1-domain and ataxin3's C-terminus. In contrast, MSP1 mutants of p97 bind ataxin3 irrespective of their nucleotide state, indicating a failure by these mutants to translate ADP binding into a movement of the N-terminal domain. Our model provides a mechanistic explanation for how nucleotides regulate the p97-ataxin3 interaction and why atypical cofactor binding is observed with MSP1 mutants. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  3. Modulation of F0F1-ATP synthase activity by cyclophilin D regulates matrix adenine nucleotide levels

    PubMed Central

    Chinopoulos, Christos; Konràd, Csaba; Kiss, Gergely; Metelkin, Eugeniy; Töröcsik, Beata; Zhang, Steven F.; Starkov, Anatoly A.

    2011-01-01

    Cyclophilin D was recently shown to bind to and decrease the activity of F0F1-ATP synthase in submitochondrial particles and permeabilized mitochondria (Giorgio et al. 2009, J Biol Chem, 284:33982). Cyclophilin D binding decreased both the ATP synthesis and hydrolysis rates. Here, we reaffirm these findings by demonstrating that in intact mouse liver mitochondria energized by ATP, absence of cyclophilin D or presence of cyclosporin A led to a decrease in the extent of uncoupler-induced depolarization. Accordingly, in substrate-energized mitochondria an increase in F0F1-ATP synthase activity mediated by a relief of inhibition by cyclophilin D was evident as slightly increased respiration rates during arsenolysis. However, the modulation of F0F1-ATP synthase by cyclophilin D did not increase the ANT-mediated ATP efflux rate in energized mitochondria or the ATP influx rate in de-energized mitochondria. The lack of effect of cyclophilin D on the ANT-mediated adenine nucleotide exchange rate was attributed to the ~2.2 times lower flux control coefficient of the F0F1-ATP synthase than that of ANT, deduced from measurements of adenine nucleotide flux rates in intact mitochondria. These findings were further supported by a recent kinetic model of the mitochondrial phosphorylation system, suggesting that a ~30% change in F0F1-ATP synthase activity in fully energized or fully deenergized mitochondria affects ADP-ATP exchange rate mediated by the ANT in the range of 1.38-1.7%. We conclude that in mitochondria exhibiting intact inner membranes, the absence of cyclophilin D or inhibition of its binding to F0F1-ATP synthase by cyclosporin A will affect only matrix adenine nucleotides levels. PMID:21281446

  4. Isolation of a gene encoding a developmentally regulated T cell-specific protein with a guanine nucleotide triphosphate-binding motif

    SciTech Connect

    Carlow, D.A.; Teh, H.S.; Marth, J.

    1995-02-15

    In this study, we describe a novel full length cDNA clone designated Tgtp that encodes a predicted 415-amino acid a T cell-specific guanine nucleotide triphosphate-binding protein (TGTP) bearing the characteristic motifs of a guanine nucleotide triphosphate (GTP) binding protein. Tgtp is expressed preferentially, if not exclusively, in T cells, and is up-regulated in both unfractionated and in purified CD4{sup +}8{sup +} thymocytes upon TCR cross-linking. In contrast, expression of Tgtp in peripheral T cells is maintained at relatively high levels and is not grossly affected by TCR cross-linking. Antiserum generated against synthetic peptides from the predicted TGTP amino acid sequence recognized a single protein with a molecular mass of {approx}50 kDa, corresponding well with the computed molecular mass of 47 kDa. The only known relative of Tgtp is MUSGTP, which is reportedly expressed in B cells and bears a GTP binding motif. Thus, the discovery of Tgtp resolves a subfamily of molecules with GTP binding motifs and apparent lymphoid lineage-restricted expression. Given the restricted expression pattern in T cells, the up-regulated expression observed in response to TCR signaling in immature thymocytes, and the presence of the motifs characteristic of GTP binding proteins, we suggest that TGTP may have an important function in T cell development and/or T cell activation. 51 refs., 6 figs.

  5. Reevaluation of pollen quantitation by an automatic pollen counter.

    PubMed

    Muradil, Mutarifu; Okamoto, Yoshitaka; Yonekura, Syuji; Chazono, Hideaki; Hisamitsu, Minako; Horiguchi, Shigetoshi; Hanazawa, Toyoyuki; Takahashi, Yukie; Yokota, Kunihiko; Okumura, Satoshi

    2010-01-01

    Accurate and detailed pollen monitoring is useful for selection of medication and for allergen avoidance in patients with allergic rhinitis. Burkard and Durham pollen samplers are commonly used, but are labor and time intensive. In contrast, automatic pollen counters allow simple real-time pollen counting; however, these instruments have difficulty in distinguishing pollen from small nonpollen airborne particles. Misidentification and underestimation rates for an automatic pollen counter were examined to improve the accuracy of the pollen count. The characteristics of the automatic pollen counter were determined in a chamber study with exposure to cedar pollens or soil grains. The cedar pollen counts were monitored in 2006 and 2007, and compared with those from a Durham sampler. The pollen counts from the automatic counter showed a good correlation (r > 0.7) with those from the Durham sampler when pollen dispersal was high, but a poor correlation (r < 0.5) when pollen dispersal was low. The new correction method, which took into account the misidentification and underestimation, improved this correlation to r > 0.7 during the pollen season. The accuracy of automatic pollen counting can be improved using a correction to include rates of underestimation and misidentification in a particular geographical area.

  6. "Plug and play" logic gates based on fluorescence switching regulated by self-assembly of nucleotide and lanthanide ions.

    PubMed

    Pu, Fang; Ren, Jinsong; Qu, Xiaogang

    2014-06-25

    Molecular logic gates in response to chemical, biological, or optical input signals at a molecular level have received much interest over the past decade. Herein, we construct "plug and play" logic systems based on the fluorescence switching of guest molecules confined in coordination polymer nanoparticles generated from nucleotide and lanthanide ions. In the system, the addition of new modules directly enables new logic functions. PASS 0, YES, PASS 1, NOT, IMP, OR, and AND gates are successfully constructed in sequence. Moreover, different logic gates (AND, INH, and IMP) can be constructed using different guest molecules and the same input combinations. The work will be beneficial to the future logic design and expand the applications of coordination polymers.

  7. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen

    PubMed Central

    Bhalla, Prem L.; Swoboda, Ines; Singh, Mohan B.

    1999-01-01

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects ≈20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible. PMID:10500236

  8. Antisense-mediated silencing of a gene encoding a major ryegrass pollen allergen.

    PubMed

    Bhalla, P L; Swoboda, I; Singh, M B

    1999-09-28

    Type 1 allergic reactions, such as hay fever and allergic asthma, triggered by grass pollen allergens are a global health problem that affects approximately 20% of the population in cool, temperate climates. Ryegrass is the dominant source of allergens because of its prodigious production of airborne pollen. Lol p 5 is the major allergenic protein of ryegrass pollen, judging from the fact that almost all of the individuals allergic to grass pollen show presence of serum IgE antibodies against this protein. Moreover, nearly two-thirds of the IgE reactivity of ryegrass pollen has been attributed to this protein. Therefore, it can be expected that down-regulation of Lol p 5 production can significantly reduce the allergic potential of ryegrass pollen. Here, we report down-regulation of Lol p 5 with an antisense construct targeted to the Lol p 5 gene in ryegrass. The expression of antisense RNA was regulated by a pollen-specific promoter. Immunoblot analysis of proteins with allergen-specific antibodies did not detect Lol p 5 in the transgenic pollen. The transgenic pollen showed remarkably reduced allergenicity as reflected by low IgE-binding capacity of pollen extract as compared with that of control pollen. The transgenic ryegrass plants in which Lol p 5 gene expression is perturbed showed normal fertile pollen development, indicating that genetic engineering of hypoallergenic grass plants is possible.

  9. Up-regulation of nucleotide excision repair in mouse lung and liver following chronic exposure to aflatoxin B{sub 1} and its dependence on p53 genotype

    SciTech Connect

    Mulder, Jeanne E.; Bondy, Genevieve S.; Mehta, Rekha; Massey, Thomas E.

    2014-03-01

    Aflatoxin B{sub 1} (AFB{sub 1}) is biotransformed in vivo into an epoxide metabolite that forms DNA adducts that may induce cancer if not repaired. p53 is a tumor suppressor gene implicated in the regulation of global nucleotide excision repair (NER). Male heterozygous p53 knockout (B6.129-Trp53{sup tm1Brd}N5, Taconic) and wild-type mice were exposed to 0, 0.2 or 1.0 ppm AFB{sub 1} for 26 weeks. NER activity was assessed with an in vitro assay, using AFB{sub 1}-epoxide adducted plasmid DNA as a substrate. For wild-type mice, repair of AFB{sub 1}–N7-Gua adducts was 124% and 96% greater in lung extracts from mice exposed to 0.2 ppm and 1.0 ppm AFB{sub 1} respectively, and 224% greater in liver extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05). In heterozygous p53 knockout mice, repair of AFB{sub 1}–N7-Gua was only 45% greater in lung extracts from mice exposed to 0.2 ppm AFB{sub 1} (p < 0.05), and no effect was observed in lung extracts from mice treated with 1.0 ppm AFB{sub 1} or in liver extracts from mice treated with either AFB{sub 1} concentration. p53 genotype did not affect basal levels of repair. AFB{sub 1} exposure did not alter repair of AFB{sub 1}-derived formamidopyrimidine adducts in lung or liver extracts of either mouse genotype nor did it affect XPA or XPB protein levels. In summary, chronic exposure to AFB{sub 1} increased NER activity in wild-type mice, and this response was diminished in heterozygous p53 knockout mice, indicating that loss of one allele of p53 limits the ability of NER to be up-regulated in response to DNA damage. - Highlights: • Mice are chronically exposed to low doses of the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}). • The effects of AFB{sub 1} and p53 status on nucleotide excision repair are investigated. • AFB{sub 1} increases nucleotide excision repair in wild type mouse lung and liver. • This increase is attenuated in p53 heterozygous mouse lung and liver. • Results portray the role of p53 in

  10. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    SciTech Connect

    Feltner, D.E.; Marasco, W.A.

    1989-06-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of (3H)FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM (3H)FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. (3H)FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of (3H)FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM (3H)FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state.

  11. The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles.

    PubMed

    Islam, Aminul; Shen, Xiaoyan; Hiroi, Toyoko; Moss, Joel; Vaughan, Martha; Levine, Stewart J

    2007-03-30

    The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

  12. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein regulates neurite development via PI3K-AKT and ERK signaling pathways.

    PubMed

    Zhou, C; Li, C; Li, D; Wang, Y; Shao, W; You, Y; Peng, J; Zhang, X; Lu, L; Shen, X

    2013-12-19

    The elongation of neuron is highly dependent on membrane trafficking. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein 1 (BIG1) functions in the membrane trafficking between the Golgi apparatus and the plasma membrane. BFA, an uncompetitive inhibitor of BIG1 can inhibit neurite outgrowth and polarity development. In this study, we aimed to define the possible role of BIG1 in neurite development and to further investigate the potential mechanism. By immunostaining, we found that BIG1 was extensively colocalized with synaptophysin, a marker for synaptic vesicles in soma and partly in neurites. The amount of both protein and mRNA of BIG1 were up-regulated during rat brain development. BIG1 depletion significantly decreased the neurite length and inhibited the phosphorylation of phosphatidylinositide 3-kinase (PI3K) and protein kinase B (AKT). Inhibition of BIG1 guanine nucleotide-exchange factor (GEF) activity by BFA or overexpression of the dominant-negative BIG1 reduced PI3K and AKT phosphorylation, indicating regulatory effects of BIG1 on PI3K-AKT signaling pathway is dependent on its GEF activity. BIG1 siRNA or BFA treatment also significantly reduced extracellular signal-regulated kinase (ERK) phosphorylation. Overexpression of wild-type BIG1 significantly increased ERK phosphorylation, but the dominant-negative BIG1 had no effect on ERK phosphorylation, indicating the involvement of BIG1 in ERK signaling regulation may not be dependent on its GEF activity. Our result identified a novel function of BIG1 in neurite development. The newly recognized function integrates the function of BIG1 in membrane trafficking with the activation of PI3K-AKT and ERK signaling pathways which are critical in neurite development. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  13. Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment

    PubMed Central

    Godinho, Rosely Oliveira; Costa-Jr, Valter Luiz

    2003-01-01

    This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid. PMID:12642402

  14. The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila

    PubMed Central

    Szul, Tomasz; Burgess, Jason; Jeon, Mili; Zinn, Kai; Marques, Guillermo; Brill, Julie A

    2011-01-01

    Surface delivery of proteins involved in cell-cell and cell-matrix interactions in cultured mammalian cells requires the GBF1 guanine nucleotide exchange factor. However, the role of GBF1 in delivery of adhesion proteins during organogenesis in intact animals has not been characterized. Here, we report the function of the fly GBF1 homolog, Gartenzwerg (Garz) in the development of the salivary gland in Drosophila melanogaster. We used the GAL4/UAS system to selectively deplete Garz from salivary gland cells. We show that depletion of Garz disrupts the secretory pathway as evidenced by the collapse of Golgi-localized Lava lamp (Lva) and the TGN-localized γ subunit of the clathrin-adaptor protein complex (AP-1). Additionally, Garz depletion inhibits trafficking of cell-cell adhesion proteins cadherin (DE-cad) and Flamingo to the cell surface. Disregulation of trafficking correlates with mistargeting of the tumor suppressor protein Discs large involved in epithelial polarity determination. Garz-depleted salivary cells are smaller and lack well-defined plasma membrane domains. Garz depletion also inhibits normal elongation and positioning of epithelial cells, resulting in a disorganized salivary gland that lacks a well defined luminal duct. Our findings suggest that Garz is essential for establishment of epithelial structures and demonstrate an absolute requirement for Garz during Drosophila development. PMID:21686256

  15. The Garz Sec7 domain guanine nucleotide exchange factor for Arf regulates salivary gland development in Drosophila.

    PubMed

    Szul, Tomasz; Burgess, Jason; Jeon, Mili; Zinn, Kai; Marques, Guillermo; Brill, Julie A; Sztul, Elizabeth

    2011-03-01

    Surface delivery of proteins involved in cell-cell and cell-matrix interactions in cultured mammalian cells requires the GBF1 guanine nucleotide exchange factor. However, the role of GBF1 in delivery of adhesion proteins during organogenesis in intact animals has not been characterized. Here, we report the function of the fly GBF1 homolog, Gartenzwerg (Garz) in the development of the salivary gland in Drosophila melanogaster. We used the GAL4/UAS system to selectively deplete Garz from salivary gland cells. We show that depletion of Garz disrupts the secretory pathway as evidenced by the collapse of Golgi-localized Lava lamp (Lva) and the TGN-localized γ subunit of the clathrin-adaptor protein complex (AP-1). Additionally, Garz depletion inhibits trafficking of cell-cell adhesion proteins cadherin (DE-cad) and Flamingo to the cell surface. Disregulation of trafficking correlates with mistargeting of the tumor suppressor protein Discs large involved in epithelial polarity determination. Garz-depleted salivary cells are smaller and lack well-defined plasma membrane domains. Garz depletion also inhibits normal elongation and positioning of epithelial cells, resulting in a disorganized salivary gland that lacks a well defined luminal duct. Our findings suggest that Garz is essential for establishment of epithelial structures and demonstrate an absolute requirement for Garz during Drosophila development.

  16. The cyclic di-nucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function

    PubMed Central

    Precit, Mimi; Delince, Matthieu; Pensinger, Daniel; Huynh, TuAnh Ngoc; Jurado, Ashley R.; Goo, Young Ah; Sadilek, Martin; Iavarone, Anthony T.; Sauer, John-Demian; Tong, Liang; Woodward, Joshua J.

    2014-01-01

    SUMMARY Cyclic di-adenosine monophosphate (c-di-AMP) is a broadly conserved second messenger required for bacterial growth and infection. However, the molecular mechanisms of c-di-AMP signaling are still poorly understood. Using a chemical proteomics screen for c-di-AMP interacting proteins in the pathogen Listeria monocytogenes, we identified several broadly conserved protein receptors, including the central metabolic enzyme pyruvate carboxylase (LmPC). Biochemical and crystallographic studies of the LmPC-c-di-AMP interaction revealed a previously unrecognized allosteric regulatory site 25 Å from the active site. Mutations in this site disrupted c-di-AMP binding and affected enzyme catalysis of LmPC as well as PC from pathogenic Enterococcus faecalis. C-di-AMP depletion resulted in altered metabolic activity in L. monocytogenes. Correction of this metabolic imbalance rescued bacterial growth, reduced bacterial lysis, and resulted in enhanced bacterial burdens during infection. These findings greatly expand the c-di-AMP signaling repertoire and reveal a central metabolic regulatory role for a cyclic di-nucleotide. PMID:25215494

  17. A MicroRNA Superfamily Regulates Nucleotide Binding Site–Leucine-Rich Repeats and Other mRNAs[W][OA

    PubMed Central

    Shivaprasad, Padubidri V.; Chen, Ho-Ming; Patel, Kanu; Bond, Donna M.; Santos, Bruno A.C.M.; Baulcombe, David C.

    2012-01-01

    Analysis of tomato (Solanum lycopersicum) small RNA data sets revealed the presence of a regulatory cascade affecting disease resistance. The initiators of the cascade are microRNA members of an unusually diverse superfamily in which miR482 and miR2118 are prominent members. Members of this superfamily are variable in sequence and abundance in different species, but all variants target the coding sequence for the P-loop motif in the mRNA sequences for disease resistance proteins with nucleotide binding site (NBS) and leucine-rich repeat (LRR) motifs. We confirm, using transient expression in Nicotiana benthamiana, that miR482 targets mRNAs for NBS-LRR disease resistance proteins with coiled-coil domains at their N terminus. The targeting causes mRNA decay and production of secondary siRNAs in a manner that depends on RNA-dependent RNA polymerase 6. At least one of these secondary siRNAs targets other mRNAs of a defense-related protein. The miR482-mediated silencing cascade is suppressed in plants infected with viruses or bacteria so that expression of mRNAs with miR482 or secondary siRNA target sequences is increased. We propose that this process allows pathogen-inducible expression of NBS-LRR proteins and that it contributes to a novel layer of defense against pathogen attack. PMID:22408077

  18. p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair

    PubMed Central

    Wang, Qi-En; Han, Chunhua; Zhao, Ran; Wani, Gulzar; Zhu, Qianzheng; Gong, Li; Battu, Aruna; Racoma, Ira; Sharma, Nidhi; Wani, Altaf A.

    2013-01-01

    Besides the primary histone acetyltransferase (HAT)-mediated chromatin remodeling function, co-transcriptional factor, p300, is also known to play a distinct role in DNA repair. However, the exact mechanism of p300 function in DNA repair has remained unclear and difficult to discern due to the phosphorylation and degradation of p300 in response to DNA damage. Here, we have demonstrated that p300 is only degraded in the presence of specific DNA lesions, which are the substrates of nucleotide excision repair (NER) pathway. In contrast, DNA double-strand breaks fail to degrade p300. Degradation is initiated by phosphorylation of p300 at serine 1834, which is catalyzed by the cooperative action of p38 mitogen-activated protein kinases and Akt kinases. In depth, functional analysis revealed that (i) p300 and CBP act redundantly in repairing ultraviolet (UV) lesions, (ii) the phosphorylation of p300 at S1834 is critical for efficient removal of UV-induced cyclobutane pyrimidine dimers and (iii) p300 is recruited to DNA damage sites located within heterochromatin. Taken together, we conclude that phosphorylated p300 initially acetylates histones to relax heterochromatin to allow damage recognition factors access to damage DNA. Thereupon, p300 is promptly degraded to allow the sequential recruitment of downstream repair proteins for successful execution of NER. PMID:23275565

  19. Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation

    PubMed Central

    Kim, Mee J.; Findlay, Gregory M.; Martin, Beth; Zhao, Jingjing; Bell, Robert J. A.; Smith, Robin P.; Ku, Angel A.; Shendure, Jay; Ahituv, Nadav

    2014-01-01

    In addition to their protein coding function, exons can also serve as transcriptional enhancers. Mutations in these exonic-enhancers (eExons) could alter both protein function and transcription. However, the functional consequence of eExon mutations is not well known. Here, using massively parallel reporter assays, we dissect the enhancer activity of three liver eExons (SORL1 exon 17, TRAF3IP2 exon 2, PPARG exon 6) at single nucleotide resolution in the mouse liver. We find that both synonymous and non-synonymous mutations have similar effects on enhancer activity and many of the deleterious mutation clusters overlap known liver-associated transcription factor binding sites. Carrying a similar massively parallel reporter assay in HeLa cells with these three eExons found differences in their mutation profiles compared to the liver, suggesting that enhancers could have distinct operating profiles in different tissues. Our results demonstrate that eExon mutations could lead to multiple phenotypes by disrupting both the protein sequence and enhancer activity and that enhancers can have distinct mutation profiles in different cell types. PMID:25340400

  20. Nucleotide sequence and functional analysis of cbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides.

    PubMed Central

    Gibson, J L; Tabita, F R

    1993-01-01

    Structural genes encoding Calvin cycle enzymes in Rhodobacter sphaeroides are duplicated and organized within two physically distinct transcriptional units, the form I and form II cbb operons. Nucleotide sequence determination of the region upstream of the form I operon revealed a divergently transcribed open reading frame, cbbR, that showed significant similarity to the LysR family of transcriptional regulatory proteins. Mutants containing an insertionally inactivated cbbR gene were impaired in photoheterotrophic growth and completely unable to grow photolithoautotrophically with CO2 as the sole carbon source. In the cbbR strain, expression of genes within the form I operon was completely abolished and that of the form II operon was reduced to about 30% of the wild-type level. The cloned cbbR gene complemented the mutant for wild-type growth characteristics, and normal levels of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) were observed. However, rocket immunoelectrophoresis revealed that the wild-type level of RubisCO was due to overexpression of the form II enzyme, whereas expression of the form I RubisCO was 10% of that of the wild-type strain. The cbbR insertional inactivation did not appear to affect aerobic expression of either CO2 fixation operon, but preliminary evidence suggests that the constitutive expression of the form II operon observed in the cbbR strain may be subject to repression during aerobic growth. PMID:8376325

  1. Digital Gene Expression Analysis of Populus simonii × P. nigra Pollen Germination and Tube Growth

    PubMed Central

    Zhao, Li-Juan; Yuan, Hong-Mei; Guo, Wen-Dong; Yang, Chuan-Ping

    2016-01-01

    Pollen tubes are an ideal model for the study of cell growth and morphogenesis because of their extreme elongation without cell division; however, the genetic basis of pollen germination and tube growth remains largely unknown. Using the Illumina/Solexa digital gene expression system, we identified 13,017 genes (representing 28.3% of the unigenes on the reference genes) at three stages, including mature pollen, hydrated pollen, and pollen tubes of Populus simonii × P. nigra. Comprehensive analysis of P. simonii × P. nigra pollen revealed dynamic changes in the transcriptome during pollen germination and pollen tube growth (PTG). Gene ontology analysis of differentially expressed genes showed that genes involved in functional categories such as catalytic activity, binding, transporter activity, and enzyme regulator activity were overrepresented during pollen germination and PTG. Some highly dynamic genes involved in pollen germination and PTG were detected by clustering analysis. Genes related to some key pathways such as the mitogen-activated protein kinase signaling pathway, regulation of the actin cytoskeleton, calcium signaling, and ubiquitin-mediated proteolysis were significantly changed during pollen germination and PTG. These data provide comprehensive molecular information toward further understanding molecular mechanisms underlying pollen germination and PTG. PMID:27379121

  2. [Nucleotide receptors and renal function].

    PubMed

    Jankowski, Maciej

    2014-01-01

    Kidney plays a key role in homeostasis of human body. It has heterogenic structure and is characterized by complicated vascular beds and numbers of sympathetic nerves endings. Nucleotides receptors are involved in the regulation of blood flow, a fundamental process for renal function. Plasma is filtrated in renal glomerulus and activity of nucleotides receptors located on cells of glomerular filter modifies the physi- cochemical properties of filter and affects the filtration process. Electrolytes, water and low molecular weight molecules are reabsorbed from tubular fluid or secreted into fluid in proximal and distal tubules. Glomerular filtration rate and activity of tubular processes are regulated via nucleotides receptors by glomerulotubularbalance and tubuloglomerular feedback. Nucleotides receptors are involved in systemic regulation of blood pressure and carbohydrate metabolism.

  3. Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells.

    PubMed

    Onetti, C G; Lara, J; García, E

    1996-05-01

    Effects of membrane potential, intracellular Ca2+ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K+ ions; the unitary conductance was 112 pS in symmetrical K+, and the K+ permeability (PK) was 1.3 x 10(-13) cm x s(-1). An inward rectification was observed when intracellular K+ was reduced. Using a quasi-physiological K+ gradient, a non-linear K+ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (Po) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K+ concentrations, the channel was activated from a threshold of about -60 mV, and the activation midpoint was -2 mV. Po decreased noticeably at 50 microM internal adenosine 5'-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 microM. Internal application of 5'-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased Po. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca2+ ions (0.1 to 10 microM) was required for the activation of this channel. The glucose--sensitive K+ channel of XO neurons could be considered as a subtype of ATP-sensitive K+ channel, contributing substantially to macroscopic outward current.

  4. Guanine Nucleotides in the Meiotic Maturation of Starfish Oocytes: Regulation of the Actin Cytoskeleton and of Ca2+ Signaling

    PubMed Central

    Kyozuka, Keiichiro; Chun, Jong T.; Puppo, Agostina; Gragnaniello, Gianni; Garante, Ezio; Santella, Luigia

    2009-01-01

    Background Starfish oocytes are arrested at the first prophase of meiosis until they are stimulated by 1-methyladenine (1-MA). The two most immediate responses to the maturation-inducing hormone are the quick release of intracellular Ca2+ and the accelerated changes of the actin cytoskeleton in the cortex. Compared with the later events of oocyte maturation such as germinal vesicle breakdown, the molecular mechanisms underlying the early events involving Ca2+ signaling and actin changes are poorly understood. Herein, we have studied the roles of G-proteins in the early stage of meiotic maturation. Methodology/Principal Findings By microinjecting starfish oocytes with nonhydrolyzable nucleotides that stabilize either active (GTPγS) or inactive (GDPβS) forms of G-proteins, we have demonstrated that: i) GTPγS induces Ca2+ release that mimics the effect of 1-MA; ii) GDPβS completely blocks 1-MA-induced Ca2+; iii) GDPβS has little effect on the amplitude of the Ca2+ peak, but significantly expedites the initial Ca2+ waves induced by InsP3 photoactivation, iv) GDPβS induces unexpectedly striking modification of the cortical actin networks, suggesting a link between the cytoskeletal change and the modulation of the Ca2+ release kinetics; v) alteration of cortical actin networks with jasplakinolide, GDPβS, or actinase E, all led to significant changes of 1-MA-induced Ca2+ signaling. Conclusions/Significance Taken together, these results indicate that G-proteins are implicated in the early events of meiotic maturation and support our previous proposal that the dynamic change of the actin cytoskeleton may play a regulatory role in modulating intracellular Ca2+ release. PMID:19617909

  5. Regulation of presynaptic terminal organization by C. elegans RPM-1, a putative guanine nucleotide exchanger with a RING-H2 finger domain.

    PubMed

    Zhen, M; Huang, X; Bamber, B; Jin, Y

    2000-05-01

    Presynaptic terminals contain highly organized subcellular structures to facilitate neurotransmitter release. In C. elegans, the typical presynaptic terminal has an electron-dense active zone surrounded by synaptic vesicles. Loss-of-function mutations in the rpm-1 gene result in abnormally structured presynaptic terminals in GABAergic neuromuscular junctions (NMJs), most often manifested as a single presynaptic terminal containing multiple active zones. The RPM-1 protein has an RCC1-like guanine nucleotide exchange factor (GEF) domain and a RING-H2 finger. RPM-1 is most similar to the Drosophila presynaptic protein Highwire (HIW) and the mammalian Myc binding protein Pam. RPM-1 is localized to the presynaptic region independent of synaptic vesicles and functions cell autonomously. The temperature-sensitive period of rpm-1 coincides with the time of synaptogenesis. rpm-1 may regulate the spatial arrangement, or restrict the formation, of presynaptic structures.

  6. Combining Electron Crystallography and X-ray Crystallography to Study the MlotiK1 Cyclic Nucleotide-Regulated Potassium Channel

    SciTech Connect

    Clayton, G.; Aller, S; Wang, J; Unger, V; Morais-Cabral, J

    2009-01-01

    We have recently reported the X-ray structure of the cyclic nucleotide-regulated potassium channel, MlotiK1. Here we describe the application of both electron and X-ray crystallography to obtain high quality crystals. We suggest that the combined application of these techniques provides a useful strategy for membrane protein structure determination. We also present negative stain projection and cryo-data projection maps. These maps provide new insights about the properties of the MlotiK1 channel. In particular, a comparison of a 9 {angstrom} cryo-data projection with calculated model maps strongly suggests that there is a very weak interaction between the pore and the S1-S4 domains of this 6 TM tetrameric cation channel and that the S1-S4 domains can adopt multiple orientations relative to the pore.

  7. IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation.

    PubMed

    Chen, Xiaoyang; Zhang, Hua; Sun, Huayue; Luo, Hongbing; Zhao, Li; Dong, Zhaobin; Yan, Shuangshuang; Zhao, Cheng; Liu, Renyi; Xu, Chunyan; Li, Song; Chen, Huabang; Jin, Weiwei

    2017-01-01

    Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize. © 2017 American Society of Plant Biologists. All Rights Reserved.

  8. Juvenile Hormone Regulates the Expression of Drosophila Epac– a Guanine Nucleotide Exchange Factor for Rap1 Small GTPase

    USDA-ARS?s Scientific Manuscript database

    The juvenile hormones (JH) are a key group of insect hormones involved in regulating larval development and adult reproductive processes. Although well-studied from the physiological standpoint, the molecular actions of JH remain unclear. Using cDNA microchip array technology, we previously identifi...

  9. Interactions between the C-terminus of Kv1.5 and Kvβ regulate pyridine nucleotide-dependent changes in channel gating

    PubMed Central

    Tipparaju, Srinivas M.; Li, Xiao-Ping; Kilfoil, Peter J.; Xue, Bin; Uversky, Vladimir N.; Bhatnagar, Aruni; Barski, Oleg A.

    2012-01-01

    Voltage-gated potassium (Kv) channels are tetrameric assemblies of transmembrane Kv proteins with cytosolic N- and C-termini. The N-terminal domain of Kv1 proteins binds to β-subunits, but the role of the C-terminus is less clear. Therefore, we studied the role of the C-terminus in regulating Kv1.5 channel and its interactions with Kvβ-subunits. When expressed in COS-7 cells, deletion of the C-terminal domain of Kv1.5 did not affect channel gating or kinetics. Co-expression of Kv1.5 with Kvβ3 increased current inactivation, whereas Kvβ2 caused a hyperpolarizing shift in the voltage-dependence of current activation. Inclusion of NADPH in the patch pipette solution accelerated the inactivation of Kv1.5-Kvβ3 currents. In contrast, NADP+ decreased the rate and the extent of Kvβ3-induced inactivation and reversed the hyperpolarizing shift in the voltage-dependence of activation induced by Kvβ2. Currents generated by Kv1.5ΔC+Kvβ3 or Kv1.5ΔC+Kvβ2 complexes did not respond to changes in intracellular pyridine nucleotide concentration, indicating that the C-terminus is required for pyridine nucleotide-dependent interactions between Kvβ and Kv1.5. A glutathione-S-transferase (GST) fusion protein containing the C-terminal peptide of Kv1.5 did not bind to apoKvβ2, but displayed higher affinity for Kvβ2:NADPH than Kvβ2:NADP+. The GST fusion protein also precipitated Kvβ proteins from mouse brain lysates. Pull-down experiments, structural analysis and electrophysiological data indicated that a specific region of the C-terminus (Arg543-Val583) is required for Kvβ binding. These results suggest that the C-terminal domain of Kv1.5 interacts with β-subunits and that this interaction is essential for the differential regulation of Kv currents by oxidized and reduced nucleotides. PMID:22426702

  10. Fructokinase and hexokinase from pollen grains of bell pepper (Capsicum annuum L.): possible role in pollen germination under conditions of high temperature and CO2 enrichment.

    PubMed

    Karni, Leah; Aloni, Beny

    2002-11-01

    The processes of pollen grain development and germination depend on the uptake and metabolism of pollen sugars. In pepper (Capsicum annuum L.), initial sugar metabolism includes sucrose hydrolysis by invertase and subsequent phosphorylation of glucose and fructose by hexose kinases. The main objective of this study was to investigate changes in fructokinase (EC 2.7.1.4) and hexokinase (EC.2.7.1.1) activities in pepper flowers during their development, and to study the possible roles of these enzymes in determining pollen germination capacity under high temperature and under CO(2) enrichment, previously shown to modify sugar concentrations in pepper pollen (Aloni et al., 2001 Physiologia Plantarum 112: 505-512). Fructokinase (FK) activity was predominant in pepper pollen, and increased during pollen maturation. Pollen hexokinase (HK) activity was low and did not change throughout pollen development. High-temperature treatment (day/night, 32/26 degrees C) of pepper plants reduced the percentage of pollen that germinated compared with that under normal temperatures (26/22 degrees C), and concomitantly reduced the activity of FK in mature pollen. High temperature also reduced FK and HK activity in the anther. Under high ambient CO(2) (800 micro l l(-1)) pollen FK activity was enhanced. The results suggest that pollen and anther FK may play a role in the regulation of pollen germination, possibly by providing fructose-6-phosphate for glycolysis, or through conversion to UDP-glucose (UDPG) to support the biosynthesis of cell wall material for pollen tube growth. High temperature stress and CO(2) enrichment may influence pollen germination capacity by affecting these pathways.

  11. Regulation of adenine nucleotide translocase and glycerol 3-phosphate dehydrogenase expression by thyroid hormones in different rat tissues.

    PubMed Central

    Dümmler, K; Müller, S; Seitz, H J

    1996-01-01

    Thyroid hormone (T3)-dependent gene expression of the adenine nucleotide translocase (ANT) and the FAD-linked glycerol 3-phosphate dehydrogenase (mGPDH) was investigated in several rat tissues. Both proteins provide an important link between cytosolic and mitochondrial metabolic pathways and seem to be involved in the stimulation of mitochondrial oxygen consumption in response to T3. Here we show that two ANT isoforms are expressed in rat, the muscle-specific ANT1 form and the ubiquitous ANT2 form. The expression of ANT1 mRNA is not sensitive to T3 whereas the amount of ANT2 mRNA is increased 7-9-fold in liver and heart within 12-48 h after T3 application. Little or no effect of T3 on ANT2 mRNA was observed in kidney and brain. The mRNA changes are paralleled by an increase in ANT protein, thus explaining the accelerated ADP/ATP exchange observed in mitochondria isolated from hyperthyroid rats. The key role of ANT2 in the control of hyperthyroid metabolism is evident because the expression of the mersalyl-sensitive phosphate carrier and the mitochondrial creatine kinase mRNA, which are functionally linked to ANT, did not respond to T3. Similarly to the ADP/ATP exchange, the transfer of cytosolic NADH to the respiratory chain via the glycerophosphate shuttle is very sensitive to T3. Recently we demonstrated the 10-15-fold induction of mGPDH mRNA in rat liver after administration of T3 [Müller and Seitz (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10581-10585]. Here we show that, in contrast with ANT2, the time course of induction is fast (4-6 h). Furthermore, mGPDH mRNA is induced 6-fold by T3 in heart and 4-fold in kidney. From these results we conclude that the T3-mediated transcriptional induction leading to increased activity of ANT2 and mGPDH contributes considerably to the increase in mitochondrial oxygen consumption in rat tissues. PMID:8760382

  12. P2Y2 Nucleotide Receptor Activation Up-regulates Vascular Cell Adhesion Molecular-1 Expression and Enhances Lymphocyte Adherence to a Human Submandibular Gland Cell Line

    PubMed Central

    Baker, Olga J.; Camden, Jean M.; Rome, Danny E.; Seye, Cheikh I.; Weisman, Gary A.

    2007-01-01

    Sjögren’s syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS. PMID:17599409

  13. P2Y2 nucleotide receptor activation up-regulates vascular cell adhesion molecule-1 [corrected] expression and enhances lymphocyte adherence to a human submandibular gland cell line.

    PubMed

    Baker, Olga J; Camden, Jean M; Rome, Danny E; Seye, Cheikh I; Weisman, Gary A

    2008-01-01

    Sjögren's syndrome (SS) is a chronic inflammatory autoimmune disease that causes salivary and lacrimal gland tissue destruction resulting in impaired secretory function. Although lymphocytic infiltration of salivary epithelium is associated with SS, the mechanisms involved have not been adequately elucidated. Our previous studies have shown that the G protein-coupled P2Y2 nucleotide receptor (P2Y2R) is up-regulated in response to damage or stress of salivary gland epithelium, and in salivary glands of the NOD.B10 mouse model of SS-like autoimmune exocrinopathy. Additionally, we have shown that P2Y2R activation up-regulates vascular cell adhesion molecule-1 (VCAM-1) expression in endothelial cells leading to the binding of monocytes. The present study demonstrates that activation of the P2Y2R in dispersed cell aggregates from rat submandibular gland (SMG) and in human submandibular gland ductal cells (HSG) up-regulates the expression of VCAM-1. Furthermore, P2Y2R activation mediated the up-regulation of VCAM-1 expression in HSG cells leading to increased adherence of lymphocytic cells. Inhibitors of EGFR phosphorylation and metalloprotease activity abolished P2Y2R-mediated VCAM-1 expression and decreased lymphocyte binding to HSG cells. Moreover, silencing of EGFR expression abolished UTP-induced VCAM-1 up-regulation in HSG cells. These results suggest that P2Y2R activation in salivary gland cells increases the EGFR-dependent expression of VCAM-1 and the binding of lymphocytes, a pathway relevant to inflammation associated with SS.

  14. Pollen Viability and Pollen Tube Attrition in Cranberry (Vaccinium macrocarpon)

    USDA-ARS?s Scientific Manuscript database

    The content of mature seed in a cranberry fruit increases with stigmatic pollen load. On average, however, only two seeds result for every tetrad of pollen deposited. What then is the fate of the two remaining pollen grains fused in each tetrad? Germination in vitro revealed that most of the grains ...

  15. [Allergenic pollens in Spain].

    PubMed

    Subiza Garrido-Lestache, J

    2004-01-01

    Allergenic pollens that cause rhinoconjuctivitis and/or asthma are those from trees or plants that pollinate through the air (anemophilic pollination) and not through insects (entomophilic pollination). Although pollen grains would seem to be too large to easily reach the intrapulmonary airways, the relationship between pollen counts and the presence of asthmatic symptoms is only too evident. This is probably because the allergens inducing seasonal asthma are not only found within pollen grains but also outside the grains in particles of less than 10 mm that are freely found in the atmosphere. The most important pollens producing pollinosis in Spain are those from cypress trees from January-March, birch trees in April (macizo galaico), Platanus hispanica (March-April), grasses and olive trees from April-June, Parietaria from April-July and Chenopodium and/or Salsola from July-September. By geographical areas, the main cause of pollinosis are grasses in the center and north of the peninsula, olive trees in the south (Jaén, Sevilla, Granada, Córdoba) and Parietaria in the Mediterranean coast (Barcelona, Murcia, Valencia).

  16. RIP1 (ROP Interactive Partner 1)/ICR1 marks pollen germination sites and may act in the ROP1 pathway in the control of polarized pollen growth.

    PubMed

    Li, Shundai; Gu, Ying; Yan, An; Lord, Elizabeth; Yang, Zhen-Biao

    2008-11-01

    Rho family small GTPases are universal signaling switches in the control of cell polarity in eukaryotic cells. Their polar distribution to the cell cortex is critical for the execution of their functions, yet the mechanism for this distribution is poorly understood. Using a yeast two-hybrid method, we identified RIP1 (ROP interactive partner 1), which belongs to a family of five members of novel proteins that share a C-terminal region that interacts with ROP. When expressed in Arabidopsis pollen, green fluorescence protein GFP-tagged RIP1 was localized to the nucleus of mature pollen. When pollen grains were hydrated in germination medium, GFP-RIP1 switched from the nucleus to the cell cortex at the future pollen germination site and was maintained in the apical cortex of germinating pollen and growing pollen tubes. RIP1 was found to interact with ROP1 in pollen tubes, and the cortical RIP1 localization was influenced by the activity of ROP1. Overexpression of RIP1 induced growth depolarization in pollen tubes, a phenotype similar to that induced by ROP1 overexpression. Interestingly, RIP1 overexpression enhanced GFP-ROP1 recruitment to the plasma membrane (PM) of pollen tubes. Based on these observations, we hypothesize that RIP1 is involved in the positive feedback regulation of ROP1 localization to the PM, leading to the establishment of a polar site for pollen germination and pollen tube growth.

  17. Latrunculin B has different effects on pollen germination and tube growth.

    PubMed Central

    Gibbon, B C; Kovar, D R; Staiger, C J

    1999-01-01

    The actin cytoskeleton is absolutely required for pollen germination and tube growth, but little is known about the regulation of actin polymer concentrations or dynamics in pollen. Here, we report that latrunculin B (LATB), a potent inhibitor of actin polymerization, had effects on pollen that were distinct from those of cytochalasin D. The equilibrium dissociation constant measured for LATB binding to maize pollen actin was determined to be 74 nM. This high affinity for pollen actin suggested that treatment of pollen with LATB would have marked effects on actin function. Indeed, LATB inhibited maize pollen germination half-maximally at 50 nM, yet it blocked pollen tube growth at one-tenth of that concentration. Low concentrations of LATB also caused partial disruption of the actin cytoskeleton in germinated maize pollen, as visualized by light microscopy and fluorescent-phalloidin staining. The amounts of filamentous actin (F-actin) in pollen were quantified by measuring phalloidin binding sites, a sensitive assay that had not been used previously for plant cells. The amount of F-actin in maize pollen increased slightly upon germination, whereas the total actin protein level did not change. LATB treatment caused a dose-dependent depolymerization of F-actin in populations of maize pollen grains and tubes. Moreover, the same concentrations of LATB caused similar depolymerization in pollen grains before germination and in pollen tubes. These data indicate that the increased sensitivity of pollen tube growth to LATB was not due to general destabilization of the actin cytoskeleton or to decreases in F-actin amounts after germination. We postulate that germination is less sensitive to LATB than tube extension because the presence of a small population of LATB-sensitive actin filaments is critical for maintenance of tip growth but not for germination of pollen, or because germination is less sensitive to partial depolymerization of the actin cytoskeleton. PMID:10590163

  18. Fast loading ester fluorescent Ca2+ and pH indicators into pollen of Pyrus pyrifolia.

    PubMed

    Qu, Haiyong; Jiang, Xueting; Shi, Zebin; Liu, Lianmei; Zhang, Shaoling

    2012-01-01

    Loading of Ca(2+)-sensitive fluorescent probes into plant cells is an essential step to measure activities of free Ca(2+) ions in cytoplasm with a fluorescent imaging technique. Fluo-3 is one of the most suitable Ca(2+) indicators for CLSM. We loaded pollen with fluo-3/AM at three different temperatures. Fluo-3/AM was successfully loaded into pollen at both low (4°C) and high (37°C) temperatures. However, high loading temperature was best suited for pollen, because germination rate of pollen and growth of pollen tubes were relatively little impaired and loading time was shortened. Moreover, Ca(2+) distribution increased in the three apertures of pollen after hydration and showed a Ca(2+) gradient, similar to the tip of growing pollen tubes. The same protocol can be used with the AM-forms of other fluorescent dyes for effective labeling. When loading BCECF-AM into pollen at high temperature, the pollen did not show a pH gradient after hydration. Ca(2+) activities and fluxes had the same periodicity as pollen germination, but pH did not show the same phase and mostly lagged behind. However, the clear zone was alkaline when pollen tube growth was slowed or stopped and turned acidic when growth recovered. It is likely that apical pH(i) regulated pollen tube growth.

  19. Overexpression of PwTUA1, a pollen-specific tubulin gene, increases pollen tube elongation by altering the distribution of α-tubulin and promoting vesicle transport

    PubMed Central

    Yu, YanLi; Li, YanZe; Li, LingLi; Lin, JinXing; Zheng, Chengchao; Zhang, LingYun

    2009-01-01

    Tubulin genes are intimately associated with cell division and cell elongation, which are central to plant secondary cell wall development. However, their roles in pollen tube polar growth remain elusive. Here, a TUA1 gene from Picea wilsonii, which is specifically expressed in pollen, was isolated. Semi-quantitative RT-PCR analysis showed that the amount of PwTUA1 transcript varied at each stage of growth of the pollen tube and was induced by calcium ions and boron. Transient expression analysis in P. wilsonii pollen indicated that PwTUA1 improved pollen germination and pollen tube growth. The pollen of transgenic Arabidopsis overexpressing PwTUA1 also showed a higher percentage of germination and faster growth than wild-type plants not only in optimal germination medium, but also in medium supplemented with elevated levels of exogenous calcium ions or boron. Immunofluorescence and electron microscopy showed α-tubulin to be enriched and more vesicles accumulated in the apex region in germinating transgenic Arabidopsis pollen compared with wild-type plants. These results demonstrate that PwTUA1 up-regulated by calcium ions and boron contributes to pollen tube elongation by altering the distribution of α-tubulin and regulating the deposition of pollen cell wall components during the process of tube growth. The possible role of PwTUA1 in microtubule dynamics and organization was discussed. PMID:19454597

  20. The bacterial cell division regulators MinD and MinC form polymers in the presence of nucleotide.

    PubMed

    Conti, Joseph; Viola, Marissa G; Camberg, Jodi L

    2015-01-16

    The Min system of proteins, consisting of MinC, MinD and MinE, is essential for normal cell division in Escherichia coli. MinC forms a polar gradient to restrict placement of the division septum to midcell. MinC localization occurs through a direct interaction with MinD, a membrane-associating Par-like ATPase. MinE stimulates ATP hydrolysis by MinD, thereby releasing MinD from the membrane. Here, we show that MinD forms polymers with MinC and ATP without the addition of phospholipids. The topological regulator MinE induces disassembly of MinCD polymers. Two MinD mutant proteins, MinD(K11A) and MinD(ΔMTS15), are unable to form polymers with MinC.

  1. ITAM Signaling by Vav Family Rho Guanine Nucleotide Exchange Factors Regulates Interstitial Transit Rates of Neutrophils In Vivo

    PubMed Central

    Mascarenhas, Francesca; Delgado, Ryan; Miller, Mark J.; Swat, Wojciech

    2009-01-01

    Background In response to infection, neutrophils are quickly recruited from the blood into inflamed tissues. The interstitial migration of neutrophils is crucial for the efficient capture and control of rapidly proliferating microbes before microbial growth can overwhelm the host's defenses. However, the molecular mechanisms that regulate interstitial migration are incompletely understood. Methodology/Principal Findings Here, we use two-photon microscopy (2PM) to study discrete steps of neutrophil responses during subcutaneous infection with bacteria. Our study demonstrates that signals emanating from ITAM-containing receptors mediated by Vav family Rho GEFs control the velocity, but not the directionality, of neutrophil migration towards sites of bacterial infection. Conclusions/Significance Here we show that during neutrophil migration towards sites of bacterial infection, signals emanating from ITAM-containing receptors specifically control interstitial neutrophil velocity. PMID:19247495

  2. (p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae*

    PubMed Central

    Oh, Young Taek; Lee, Kang-Mu; Bari, Wasimul; Raskin, David M.; Yoon, Sang Sun

    2015-01-01

    When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ΔrelAΔrelV and ΔrelAΔrelVΔspoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ΔrelAΔspoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp0 mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ΔrelAΔspoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp0 mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine. PMID:25882848

  3. Ontogenetic development of the striatal [3H]spiperone binding: regulation by sodium and guanine nucleotide in rats.

    PubMed

    Nomura, Y; Oki, K; Segawa, T

    1982-04-01

    Ontogenetic development of specific [3H]spiperone binding to crude synaptic membranes and its regulation by Na+ and GTP was investigated in the rat striatum. (d)-Butaclamol more effectively inhibited [3H]spiperone binding than (l)-butaclamol. The ratio of inhibitory activity of (d)- and (l)-butaclamol for [3H]spiperone binding was not different between 1-, 7-, and 70-day-old animals but eight- to ninefold lower at 18 days of gestation than during the postnatal period. A Scatchard plot of specific binding indicated the presence of two types of binding: low-affinity (KD = 1.51 nM) and high-affinity (KD = 0.09 nM) binding on day 70. Only one component (KD = 0.075 nM) was observed on days 1 and 7 and both types of binding were found on day 15. Bmax gradually increased with age and reached a peak on day 30, followed by a decline on days 70 and 360. Na+, 100 mM, significantly increased specific binding on days 1, 7, 15, and 70. GTP, 50 microM, completely reversed the Na+-induced decrease in IC50 of apomorphine on both days 15 and 70, but not on day 7. It is suggested that receptors could recognize ligand stereospecificity on day 1. The density in dopamine receptors in the striatum reaches a peak on day 30, followed by a decrease on days 70 and 360. In addition, regulation by Na+ and GTP in agonist binding to dopamine receptors seems to become functional between 1 and 2 weeks after birth.

  4. Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis.

    PubMed

    Gross, Christian H; Abdul-Manan, Norzehan; Fulghum, John; Lippke, Judith; Liu, Xun; Prabhakar, Prakash; Brennan, Debra; Willis, Melissa Swope; Faerman, Carlos; Connelly, Patrick; Raybuck, Scott; Moore, Jonathan

    2006-02-17

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.

  5. Phosphorylation and activation of hamster carbamyl phosphate synthetase II by cAMP-dependent protein kinase. A novel mechanism for regulation of pyrimidine nucleotide biosynthesis.

    PubMed Central

    Carrey, E A; Campbell, D G; Hardie, D G

    1985-01-01

    The trifunctional protein CAD, which contains the first three enzyme activities of pyrimidine nucleotide biosynthesis (carbamyl phosphate synthetase II, aspartate transcarbamylase and dihydro-orotase), is phosphorylated stoichiometrically by cyclic AMP-dependent protein kinase. Phosphorylation activates the ammonia-dependent carbamyl phosphate synthetase activity of the complex by reducing the apparent Km for ATP. This effect is particularly marked in the presence of the allosteric feedback inhibitor, UTP, when the apparent Km is reduced by greater than 4-fold. Inhibition by physiological concentrations of UTP is substantially relieved by phosphorylation. Cyclic AMP-dependent protein kinase phosphorylates two serine residues on the protein termed sites 1 and 2, and the primary structures of tryptic peptides containing these sites have been determined: Site 1: Arg-Leu-Ser(P)-Ser-Phe-Val-Thr-Lys Site 2: Ile-His-Arg-Ala-Ser(P)-Asp-Pro-Gly-Leu-Pro-Ala-Glu-Glu-Pro-Lys During the phosphorylation reaction, activation of the carbamyl phosphate synthetase shows a better correlation with occupancy of site 1 rather than site 2. Both phosphorylation and activation can be reversed using purified preparations of the catalytic subunits of protein phosphatases 1- and -2A, and inactivation also correlates better with dephosphorylation of site 1 rather than site 2. We believe this to be the first report that a key enzyme in nucleotide biosynthesis is regulated in a significant manner by reversible covalent modification. The physiological role of this phosphorylation in the stimulation of cell proliferation by growth factors and other mitogens is discussed. Images Fig. 1. Fig. 5. PMID:4092695

  6. Hybridizing pines with diluted pollen

    Treesearch

    Robert Z. Callaham

    1967-01-01

    Diluted pollens would have many uses by the tree breeder. Dilutions would be particularly advantageous in making many controlled pollinations with a limited amount of pollen. They also would be useful in artificial mass pollinations of orchards or single trees. Diluted pollens might help overcome troublesome genetic barriers to crossing. Feasibility o,f using diluted...

  7. Functional expression and regulation of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in mouse iPS cell-derived cardiomyocytes after UTF1 -neo selection.

    PubMed

    Semmler, Judith; Lehmann, Martin; Pfannkuche, Kurt; Reppel, Michael; Hescheler, Jürgen; Nguemo, Filomain

    2014-01-01

    In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and If currents as well as functional hormonal regulation and sensitivity to If channel blockers. We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes. © 2014 S. Karger AG, Basel.

  8. BIG1, a brefeldin A-inhibited guanine nucleotide-exchange factor, is required for GABA-gated Cl⁻ influx through regulation of GABAA receptor trafficking.

    PubMed

    Li, Cuixian; Chen, Shaorui; Yu, Yang; Zhou, Chun; Wang, Ying; Le, Kang; Li, Dong; Shao, Weiwei; Lu, Liang; You, Yan; Peng, Jin; Huang, Heqing; Liu, Peiqing; Shen, Xiaoyan

    2014-04-01

    GABAA receptors (GABAARs) mediate the majority of fast synaptic inhibition. Trafficking regulation and protein-protein interactions that maintain the appropriate number of GABAARs at the cell surface are considered to be important mechanisms for controlling the strength of synaptic inhibition. Here, we report that BIG1, a brefeldin A (BFA)-inhibited guanine nucleotide-exchange factor (GEF) which has a known role in vesicle trafficking, is a new binding partner of GABAARs. Treatment of neurons with BFA, an uncompetitive inhibitor of BIG1 GEF activity, or depletion of BIG1 by small RNA interference (siRNA) significantly decreased GABAARs at the neuronal surface and suppressed GABA-gated influx of chloride ions. Over-expression of HA-tagged BIG1-E793K, a dominant-negative mutant, also significantly decreased GABAARs at the neuronal surface, but had no effect on the total amount of GABAARs. Inhibition of GABAAR endocytosis by muscimol increased both GABAARs and BIG1 at the neuronal surface in a time-dependent fashion, and this increase could be abolished by bicuculline. Finally, depletion of BIG1 by siRNA inhibited the muscimol-stimulated increase of GABAARs. Those data suggest an important function of BIG1 in trafficking of GABAARs to the cell surface through its GEF activity. Thus, we identify an important role of BIG1 in modulating GABA-gated Cl(-) influx through the regulation of cell surface expression of GABAARs.

  9. Dock6, a Dock-C subfamily guanine nucleotide exchanger, has the dual specificity for Rac1 and Cdc42 and regulates neurite outgrowth.

    PubMed

    Miyamoto, Yuki; Yamauchi, Junji; Sanbe, Atsushi; Tanoue, Akito

    2007-02-15

    Small GTPases of the Rho family, Rho, Rac, and Cdc42, are critical regulators of the changes in the actin cytoskeleton. Rho GTPases are typically activated by Dbl-homology (DH)-domain-containing guanine nucleotide exchange factors (GEFs). Recent genetic and biochemical studies revealed a new type of GEF for the Rho GTPases. This family is composed of 11 genes, designated as Dock1 to Dock11, and is structurally divided into four classes Dock-A, -B, -C, and -D. Dock-A and -B subfamilies are typically GEFs specific for Rac1, while the Dock-D subfamily is specific for Cdc42. Here we show that Dock6, a member of the Dock-C subfamily, exchanges GDP for GTP for Rac1 and Cdc42 in vitro and in vivo. Furthermore, we find that, in mouse N1E-115 neuroblastoma cells, expression of Dock6 is increased following differentiation. Transfection of the catalytic Dock Homology Region-2 (DHR-2) domain of Dock6 promotes neurite outgrowth mediated by Rac1 and Cdc42. Conversely, knockdown of endogenous Dock6 by small interference RNA reduces activation of Rac1 and Cdc42 and neurite outgrowth. Taken together, these results suggest that Dock6 differs from all of the identified Dock180-related proteins, in that it is the GEF specific for both Rac1 and Cdc42 and may be one of physiological regulators of neurite outgrowth.

  10. The H-loop in the Second Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator is Required for Efficient Chloride Channel Closing

    PubMed Central

    Kloch, Monika; Milewski, Michał; Nurowska, Ewa; Dworakowska, Beata; Cutting, Garry R.; Dołowy, Krzysztof

    2010-01-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a cAMP-activated chloride channel. The recent model of CFTR gating predicts that the ATP binding to both nucleotide-binding domains (NBD1 and NBD2) of CFTR is required for the opening of the channel, while the ATP hydrolysis at NBD2 induces subsequent channel closing. In most ABC proteins, efficient hydrolysis of ATP requires the presence of the invariant histidine residue within the H-loop located in the C-terminal part of the NBD. However, the contribution of the corresponding region (H-loop) of NBD2 to the CFTR channel gating has not been examined so far. Here we report that the alanine substitution of the conserved dipeptide HR motif (HR→AA) in the H-loop of NBD2 leads to prolonged open states of CFTR channel, indicating that the H-loop is required for efficient channel closing. On the other hand, the HR→AA substitution lead to the substantial decrease of CFTR-mediated current density (pA/pF) in transfected HEK 293 cells, as recorded in the whole-cell patch-clamp analysis. These results suggest that the H-loop of NBD2, apart from being required for CFTR channel closing, may be involved in regulating CFTR trafficking to the cell surface. PMID:20110677

  11. Nucleotide sequencing and characterization of Pseudomonas putida catR: a positive regulator of the catBC operon is a member of the LysR family.

    PubMed Central

    Rothmel, R K; Aldrich, T L; Houghton, J E; Coco, W M; Ornston, L N; Chakrabarty, A M

    1990-01-01

    Pseudomonas putida utilizes the catBC operon for growth on benzoate as a sole carbon source. This operon is positively regulated by the CatR protein, which is encoded from a gene divergently oriented from the catBC operon. The catR gene encodes a 32.2-kilodalton polypeptide that binds to the catBC promoter region in the presence or absence of the inducer cis-cis-muconate, as shown by gel retardation studies. However, the inducer is required for transcriptional activation of the catBC operon. The catR promoter has been localized to a 385-base-pair fragment by using the broad-host-range promoter-probe vector pKT240. This fragment also contains the catBC promoter whose -35 site is separated by only 36 nucleotides from the predicted CatR translational start. Dot blot analysis suggests that CatR binding to this dual promoter-control region, in addition to inducing the catBC operon, may also regulate its own expression. Data from a computer homology search using the predicted amino acid sequence of CatR, deduced from the DNA sequence, showed CatR to be a member of a large class of procaryotic regulatory proteins designated the LysR family. Striking homology was seen between CatR and a putative regulatory protein, TfdS. Images FIG. 4 FIG. 5 FIG. 6 PMID:1688844

  12. (p)ppGpp, a Small Nucleotide Regulator, Directs the Metabolic Fate of Glucose in Vibrio cholerae.

    PubMed

    Oh, Young Taek; Lee, Kang-Mu; Bari, Wasimul; Raskin, David M; Yoon, Sang Sun

    2015-05-22

    When V. cholerae encounters nutritional stress, it activates (p)ppGpp-mediated stringent response. The genes relA and relV are involved in the production of (p)ppGpp, whereas the spoT gene encodes an enzyme that hydrolyzes it. Herein, we show that the bacterial capability to produce (p)ppGpp plays an essential role in glucose metabolism. The V. cholerae mutants defective in (p)ppGpp production (i.e. ΔrelAΔrelV and ΔrelAΔrelVΔspoT mutants) lost their viability because of uncontrolled production of organic acids, when grown with extra glucose. In contrast, the ΔrelAΔspoT mutant, a (p)ppGpp overproducer strain, exhibited better growth in the presence of the same glucose concentration. An RNA sequencing analysis demonstrated that transcriptions of genes consisting of an operon for acetoin biosynthesis were markedly elevated in N16961, a seventh pandemic O1 strain, but not in its (p)ppGpp(0) mutant during glucose-stimulated growth. Transposon insertion in acetoin biosynthesis gene cluster resulted in glucose-induced loss of viability of the ΔrelAΔspoT mutant, further suggesting the crucial role of acetoin production in balanced growth under glucose-rich environments. Additional deletion of the aphA gene, encoding a negative regulator for acetoin production, failed to rescue the (p)ppGpp(0) mutant from the defective glucose-mediated growth, suggesting that (p)ppGpp-mediated acetoin production occurs independent of the presence of AphA. Overall, our results reveal that (p)ppGpp, in addition to its well known role as a stringent response mediator, positively regulates acetoin production that contributes to the successful glucose metabolism and consequently the proliferation of V. cholerae cells under a glucose-rich environment, a condition that may mimic the human intestine. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. GTPase of the Immune-Associated Nucleotide Protein 5 Regulates the Lysosomal Calcium Compartment in T Lymphocytes

    PubMed Central

    Serrano, Daniel; Ghobadi, Farnaz; Boulay, Guylain; Ilangumaran, Subburaj; Lavoie, Christine; Ramanathan, Sheela

    2017-01-01

    T lymphocytes from Gimap5lyp/lyp rats carrying a recessive mutation in the GTPase of immune-associated protein 5 (Gimap5) gene undergo spontaneous apoptosis. Molecular mechanisms underlying this survival defect are not yet clear. We have shown that Gimap5lyp/lyp T lymphocytes display reduced calcium influx following T cell antigen receptor (TCR) stimulation that was associated with impaired buffering of calcium by mitochondria. Here, we investigated the subcellular localization of GIMAP5 and its influence on Ca2+ response in HEK293T cells and T lymphocytes. The more abundantly expressed GIMAP5v2 localizes to the lysosome and certain endosomal vesicles. Gimap5lyp/lyp T lymphocytes showed increased accumulation of calcium in the lysosomes as evidenced by Gly-Phe β-naphthylamide (GPN) triggered Ca2+ release. As a corollary, GPN-induced Ca2+ flux was decreased in HEK293T cells expressing GIMAP5v2. Strikingly, TCR stimulation of rat, mouse, and human T lymphocytes increased lysosomal calcium content. Overall, our findings show that lysosomes modulate cellular Ca2+ response during T cell activation and that GIMAP5 regulates the lysosomal Ca2+ compartment in T lymphocytes. PMID:28223986

  14. Up-regulation and activation of the P2Y(2) nucleotide receptor mediate neurite extension in IL-1β-treated mouse primary cortical neurons.

    PubMed

    Peterson, Troy S; Thebeau, Christina N; Ajit, Deepa; Camden, Jean M; Woods, Lucas T; Wood, W Gibson; Petris, Michael J; Sun, Grace Y; Erb, Laurie; Weisman, Gary A

    2013-06-01

    The pro-inflammatory cytokine interleukin-1β (IL-1β), whose levels are elevated in the brain in Alzheimer's and other neurodegenerative diseases, has been shown to have both detrimental and beneficial effects on disease progression. In this article, we demonstrate that incubation of mouse primary cortical neurons (mPCNs) with IL-1β increases the expression of the P2Y2 nucleotide receptor (P2Y2R) and that activation of the up-regulated receptor with UTP, a relatively selective agonist of the P2Y2R, increases neurite outgrowth. Consistent with the accepted role of cofilin in the regulation of neurite extension, results indicate that incubation of IL-1β-treated mPCNs with UTP increases the phosphorylation of cofilin, a response absent in PCNs isolated from P2Y2R(-/-) mice. Other findings indicate that function-blocking anti-αv β3/5 integrin antibodies prevent UTP-induced cofilin activation in IL-1β-treated mPCNs, suggesting that established P2Y2R/αv β3/5 interactions that promote G12 -dependent Rho activation lead to cofilin phosphorylation involved in neurite extension. Cofilin phosphorylation induced by UTP in IL-1β-treated mPCNs is also decreased by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), suggesting a role for P2Y2R-mediated and Gq-dependent calcium mobilization in neurite outgrowth. Taken together, these studies indicate that up-regulation of P2Y2Rs in mPCNs under pro-inflammatory conditions can promote cofilin-dependent neurite outgrowth, a neuroprotective response that may be a novel pharmacological target in the treatment of neurodegenerative diseases. © 2013 International Society for Neurochemistry.

  15. Regulation and Function of the Nucleotide Binding Domain Leucine-Rich Repeat-Containing Receptor, Pyrin Domain-Containing-3 Inflammasome in Lung Disease

    PubMed Central

    Lee, Seonmin; Suh, Gee-Young; Ryter, Stefan W.

    2016-01-01

    Inflammasomes are specialized inflammatory signaling platforms that govern the maturation and secretion of proinflammatory cytokines, such as IL-1β and IL-18, through the regulation of caspase-1–dependent proteolytic processing. Several nucleotide binding domain leucine-rich repeat-containing receptor (NLR) family members (i.e., NLR family, pyrin domain containing [NLRP] 1, NLRP3, and NLR family, caspase recruitment domain containing-4 [NLRC4]) as well as the pyrin and hemopoietic expression, interferon-inducibility, nuclear localization domain–containing family member, absent in melanoma 2, can form inflammasome complexes in human cells. In particular, the NLRP3 inflammasome is activated in response to cellular stresses through a two-component pathway, involving Toll-like receptor 4–ligand interaction (priming) followed by a second signal, such as ATP-dependent P2X purinoreceptor 7 receptor activation. Emerging studies suggest that the NLRP3 inflammasome can exert pleiotropic effects in human diseases with potentially both pro- and antipathogenic sequelae. Whereas NLRP3 inflammasome activation can serve as a vital component of host defense against invading bacteria and pathogens, excessive activation of the inflammasome can lead to inflammation-associated tissue injury in the setting of chronic disease. In addition, pyroptosis, an inflammasome-associated mode of cell death, contributes to host defense. Recent research has described the regulation and function of the NLRP3 inflammasome in various pulmonary diseases, including acute lung injury and acute respiratory distress syndrome, sepsis, respiratory infections, chronic obstructive pulmonary disease, asthma, pulmonary hypertension, cystic fibrosis, and idiopathic pulmonary fibrosis. The NLRP3 and related inflammasomes, and their regulated cytokines or receptors, may represent novel diagnostic or therapeutic targets in pulmonary diseases and other diseases in which inflammation contributes to pathogenesis

  16. Retinoic acid-induced gene-I (RIG-I) associates with nucleotide-binding oligomerization domain-2 (NOD2) to negatively regulate inflammatory signaling.

    PubMed

    Morosky, Stefanie A; Zhu, Jianzhong; Mukherjee, Amitava; Sarkar, Saumendra N; Coyne, Carolyn B

    2011-08-12

    Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling.

  17. Retinoic Acid-induced Gene-I (RIG-I) Associates with Nucleotide-binding Oligomerization Domain-2 (NOD2) to Negatively Regulate Inflammatory Signaling*

    PubMed Central

    Morosky, Stefanie A.; Zhu, Jianzhong; Mukherjee, Amitava; Sarkar, Saumendra N.; Coyne, Carolyn B.

    2011-01-01

    Cytoplasmic caspase recruiting domain (CARD)-containing molecules often function in the induction of potent antimicrobial responses in order to protect mammalian cells from invading pathogens. Retinoic acid-induced gene-I (RIG-I) and nucleotide binding oligomerization domain 2 (NOD2) serve as key factors in the detection of viral and bacterial pathogens, and in the subsequent initiation of innate immune signals to combat infection. RIG-I and NOD2 share striking similarities in their cellular localization, both localize to membrane ruffles in non-polarized epithelial cells and both exhibit a close association with the junctional complex of polarized epithelia. Here we show that RIG-I and NOD2 not only colocalize to cellular ruffles and cell-cell junctions, but that they also form a direct interaction that is mediated by the CARDs of RIG-I and multiple regions of NOD2. Moreover, we show that RIG-I negatively regulates ligand-induced nuclear factor-κB (NF-κB) signaling mediated by NOD2, and that NOD2 negatively regulates type I interferon induction by RIG-I. We also show that the three main Crohn disease-associated mutants of NOD2 (1007fs, R702W, G908R) form an interaction with RIG-I and negatively regulate its signaling to a greater extent than wild-type NOD2. Our results show that in addition to their role in innate immune recognition, RIG-I and NOD2 form a direct interaction at actin-enriched sites within cells and suggest that this interaction may impact RIG-I- and NOD2-dependent innate immune signaling. PMID:21690088

  18. Emerging pollen allergens.

    PubMed

    Rodríguez, Rosalía; Villalba, Mayte; Batanero, Eva; Palomares, Oscar; Salamanca, Guillermo

    2007-01-01

    Numerous pollen allergens have been reported over the last few years. Most of them belong to well-known families of proteins but some others constitute the first member of new allergenic families. Some of the factors that can contribute to the detection and identification of new pollen allergens are: a) advances in the technology tools for molecular analysis; and b) the deep knowledge of many allergenic sources. The combination of these factors has provided vast information on the olive pollen allergogram and the identification of minor allergens that become major ones for a significant population. The close taxonomical relationship between olive tree and ash -both Oleaceae- has permitted to identify Fra e 1 (the Ole e 1-like allergen) in ash pollen and to detect the presence of protein homologues of Ole e 3 and Ole e 6. In the other hand, extensive areas of south Europe are suffering an increasing desertification. As a consequence of this, new botanical species are spontaneously growing in these areas or being used in greening ground programs: Chenopodium album and Salsola kali are some examples recently recognized as allergenic woods. The identification of the complete panel of allergens from the hypersensitizing sources might help to develop more accurate diagnosis, and efficient and safer therapy tools for Type-I allergic diseases.

  19. Flavonoids, alkali earth and rare earth elements affect germination of pecan pollen

    USDA-ARS?s Scientific Manuscript database

    The factors regulating pecan [Carya illinoinensis (Wangenh.) K. Koch] pollen grain germination on receptive stigmatic flower surfaces in vivo or in vitro in pollen viability assays are poorly understood. While there are many potential regulating factors, there is evidence for involvement of flavonol...

  20. The LuWD40-1 gene encoding WD repeat protein regulates growth and pollen viability in flax (Linum Usitatissimum L.).

    PubMed

    Kumar, Santosh; Jordan, Mark C; Datla, Raju; Cloutier, Sylvie

    2013-01-01

    As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem) while the transcript levels declined during reproductive development (ovary, anthers) and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax.

  1. Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

    PubMed Central

    Zhou, E M; Kisil, F T

    1995-01-01

    A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen. PMID:7751014

  2. Regulation of levels of serum antibodies to ryegrass pollen allergen Lol pIV by an internal image anti-idiotypic monoclonal antibody.

    PubMed

    Zhou, E M; Kisil, F T

    1995-03-01

    A murine monoclonal anti-idiotypic antibody (anti-Id), designated B1/1, was produced against an idiotope of a murine antibody (mAb91), which recognizes the epitope, site A, of allergen Lol pIV, one of the major groups of allergens in ryegrass (Lolium perenne) pollen. The ability of B1/1 to modulate the antibody responses to Lol pIV was investigated in murine model systems. In the first system, B1/1-keyhole limpet haemocyanin (KLH) conjugate was administered to treat three different strains of mice (C57BL/6, BALB/c and C3H). In the second and third model systems, a solution of B1/1 in phosphate-buffered saline (PBS) was used to treat syngeneic BALB/c mice at various doses and time intervals, respectively. The treatment with either form of B1/1, administered at doses ranging from 100 ng to 100 micrograms mouse, resulted in a reduction of the levels of the antibodies to Lol pIV. In particular, the level of IgE antibodies to Lol pIV was greatly reduced. The administration of a single intravenous (i.v.) injection of a solution of B1/1 8 weeks prior to the challenge with Lol pIV was still effective in reducing the level of antibodies to the allergen. Moreover, the level of antibodies to Lol pIV that expressed the idiotope mAb91 was also markedly decreased. By contrast, it was observed that the level of antibodies to Lol pIV in mice pretreated with B1/1 in PBS at a dose of 10 ng/mouse increased (albeit slightly) compared to that in mice treated with control mAb. These experimental models lend themselves for investigating the mechanism(s) by which an anti-Id modulates antibody responses to a grass pollen allergen.

  3. The LuWD40-1 Gene Encoding WD Repeat Protein Regulates Growth and Pollen Viability in Flax (Linum Usitatissimum L.)

    PubMed Central

    Kumar, Santosh; Jordan, Mark C.; Datla, Raju; Cloutier, Sylvie

    2013-01-01

    As a crop, flax holds significant commercial value for its omega-3 rich oilseeds and stem fibres. Canada is the largest producer of linseed but there exists scope for significant yield improvements. Implementation of mechanisms such as male sterility can permit the development of hybrids to assist in achieving this goal. Temperature sensitive male sterility has been reported in flax but the leakiness of this system in field conditions limits the production of quality hybrid seeds. Here, we characterized a 2,588 bp transcript differentially expressed in male sterile lines of flax. The twelve intron gene predicted to encode a 368 amino acid protein has five WD40 repeats which, in silico, form a propeller structure with putative nucleic acid and histone binding capabilities. The LuWD40-1 protein localized to the nucleus and its expression increased during the transition and continued through the vegetative stages (seed, etiolated seedling, stem) while the transcript levels declined during reproductive development (ovary, anthers) and embryonic morphogenesis of male fertile plants. Knockout lines for LuWD40-1 in flax failed to develop shoots while overexpression lines showed delayed growth phenotype and were male sterile. The non-viable flowers failed to open and the pollen grains from these flowers were empty. Three independent transgenic lines overexpressing the LuWD40-1 gene had ∼80% non-viable pollen, reduced branching, delayed flowering and maturity compared to male fertile genotypes. The present study provides new insights into a male sterility mechanism present in flax. PMID:23935935

  4. Intersectin 1L Guanine Nucleotide Exchange Activity Is Regulated by Adjacent src Homology 3 Domains That Are Also Involved in Endocytosis

    PubMed Central

    Zamanian, Jennifer L.; Kelly, Regis B.

    2003-01-01

    Intersectin 1L is a scaffolding protein involved in endocytosis that also has guanine nucleotide exchange activity for Cdc42. In the context of the full-length protein, the catalytic exchange activity of the DH domain is repressed. Here we use biochemical methods to dissect the mechanism for this inhibition. We demonstrate that the intersectin 1L SH3 domains, which bind endocytic proteins, directly inhibit the activity of the DH domain in assays for both binding and exchange of Cdc42. This inhibitory mechanism seems to act through steric hindrance of Cdc42 binding by an intramolecular interaction between the intersectin 1L SH3 domain region and the adjacent DH domain. Surprisingly, the mode of SH3 domain binding is other than through the proline peptide binding pocket. The dual role of the SH3 domains in endocytosis and repression of exchange activity suggests that the intersectin 1L exchange activity is regulated by endocytosis. We show that the endocytic protein, dynamin, competes for binding to the SH3 domains with the neural Wiskott-Aldrich Syndrome protein, an actin filament nucleation protein that is a substrate for activated Cdc42. Swapping of SH3 domain binding partners might act as a switch controlling the actin nucleation activity of intersectin 1L. PMID:12686614

  5. C(2)-ceramide influences the expression and insulin-mediated regulation of cyclic nucleotide phosphodiesterase 3B and lipolysis in 3T3-L1 adipocytes.

    PubMed

    Mei, Jie; Holst, Lena Stenson; Landström, Tova Rahn; Holm, Cecilia; Brindley, David; Manganiello, Vincent; Degerman, Eva

    2002-03-01

    Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.

  6. Stabilization of a nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator yields insight into disease-causing mutations.

    PubMed

    Vernon, Robert M; Chong, P Andrew; Lin, Hong; Yang, Zhengrong; Zhou, Qingxian; Aleksandrov, Andrei A; Dawson, Jennifer E; Riordan, John R; Brouillette, Christie G; Thibodeau, Patrick H; Forman-Kay, Julie D

    2017-08-25

    Characterization of the second nucleotide-binding domain (NBD2) of the cystic fibrosis transmembrane conductance regulator (CFTR) has lagged behind research into the NBD1 domain, in part because NBD1 contains the F508del mutation, which is the dominant cause of cystic fibrosis. Research on NBD2 has also been hampered by the overall instability of the domain and the difficulty of producing reagents. Nonetheless, multiple disease-causing mutations reside in NBD2, and the domain is critical for CFTR function, because channel gating involves NBD1/NBD2 dimerization, and NBD2 contains the catalytically active ATPase site in CFTR. Recognizing the paucity of structural and biophysical data on NBD2, here we have defined a bioinformatics-based method for manually identifying stabilizing substitutions in NBD2, and we used an iterative process of screening single substitutions against thermal melting points to both produce minimally mutated stable constructs and individually characterize mutations. We present a range of stable constructs with minimal mutations to help inform further research on NBD2. We have used this stabilized background to study the effects of NBD2 mutations identified in cystic fibrosis (CF) patients, demonstrating that mutants such as N1303K and G1349D are characterized by lower stability, as shown previously for some NBD1 mutations, suggesting a potential role for NBD2 instability in the pathology of CF. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Ubiquitin Regulates Caspase Recruitment Domain-mediated Signaling by Nucleotide-binding Oligomerization Domain-containing Proteins NOD1 and NOD2*

    PubMed Central

    Ver Heul, Aaron M.; Fowler, C. Andrew; Ramaswamy, S.; Piper, Robert C.

    2013-01-01

    NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2. PMID:23300079

  8. Ubiquitin regulates caspase recruitment domain-mediated signaling by nucleotide-binding oligomerization domain-containing proteins NOD1 and NOD2.

    PubMed

    Ver Heul, Aaron M; Fowler, C Andrew; Ramaswamy, S; Piper, Robert C

    2013-03-08

    NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins) are intracellular pattern recognition receptors that activate inflammation and autophagy. These pathways rely on the caspase recruitment domains (CARDs) within the receptors, which serve as protein interaction platforms that coordinately regulate immune signaling. We show that NOD1 CARD binds ubiquitin (Ub), in addition to directly binding its downstream targets receptor-interacting protein kinase 2 (RIP2) and autophagy-related protein 16-1 (ATG16L1). NMR spectroscopy and structure-guided mutagenesis identified a small hydrophobic surface of NOD1 CARD that binds Ub. In vitro, Ub competes with RIP2 for association with NOD1 CARD. In vivo, we found that the ligand-stimulated activity of NOD1 with a mutant CARD lacking Ub binding but retaining ATG16L1 and RIP2 binding is increased relative to wild-type NOD1. Likewise, point mutations in the tandem NOD2 CARDs at positions analogous to the surface residues defining the Ub interface on NOD1 resulted in loss of Ub binding and increased ligand-stimulated NOD2 signaling. These data suggest that Ub binding provides a negative feedback loop upon NOD-dependent activation of RIP2.

  9. Architecture of the eIF2B regulatory subcomplex and its implications for the regulation of guanine nucleotide exchange on eIF2

    PubMed Central

    Kuhle, Bernhard; Eulig, Nora K.; Ficner, Ralf

    2015-01-01

    Eukaryal translation initiation factor 2B (eIF2B) acts as guanine nucleotide exchange factor (GEF) for eIF2 and forms a central target for pathways regulating global protein synthesis. eIF2B consists of five non-identical subunits (α–ϵ), which assemble into a catalytic subcomplex (γ, ϵ) responsible for the GEF activity, and a regulatory subcomplex (α, β, δ) which regulates the GEF activity under stress conditions. Here, we provide new structural and functional insight into the regulatory subcomplex of eIF2B (eIF2BRSC). We report the crystal structures of eIF2Bβ and eIF2Bδ from Chaetomium thermophilum as well as the crystal structure of their tetrameric eIF2B(βδ)2 complex. Combined with mutational and biochemical data, we show that eIF2BRSC exists as a hexamer in solution, consisting of two eIF2Bβδ heterodimers and one eIF2Bα2 homodimer, which is homologous to homohexameric ribose 1,5-bisphosphate isomerases. This homology is further substantiated by the finding that eIF2Bα specifically binds AMP and GMP as ligands. Based on our data, we propose a model for eIF2BRSC and its interactions with eIF2 that is consistent with previous biochemical and genetic data and provides a framework to better understand eIF2B function, the molecular basis for Gcn−, Gcd− and VWM/CACH mutations and the evolutionary history of the eIF2B complex. PMID:26384431

  10. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    SciTech Connect

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  11. Impact of the [delta]F508 Mutation in First Nucleotide-binding Domain of Human Cystic Fibrosis Transmembrane Conductance Regulator on Domain Folding and Structure

    SciTech Connect

    Lewis, Hal A.; Zhao, Xun; Wang, Chi; Sauder, J. Michael; Rooney, Isabelle; Noland, Brian W.; Lorimer, Don; Kearins, Margaret C.; Conners, Kris; Condon, Brad; Maloney, Peter C.; Guggino, William B.; Hunt, John F.; Emtage, Spencer

    2010-07-19

    Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator (CFTR), commonly the deletion of residue Phe-508 (DeltaF508) in the first nucleotide-binding domain (NBD1), which results in a severe reduction in the population of functional channels at the epithelial cell surface. Previous studies employing incomplete NBD1 domains have attributed this to aberrant folding of DeltaF508 NBD1. We report structural and biophysical studies on complete human NBD1 domains, which fail to demonstrate significant changes of in vitro stability or folding kinetics in the presence or absence of the DeltaF508 mutation. Crystal structures show minimal changes in protein conformation but substantial changes in local surface topography at the site of the mutation, which is located in the region of NBD1 believed to interact with the first membrane spanning domain of CFTR. These results raise the possibility that the primary effect of DeltaF508 is a disruption of proper interdomain interactions at this site in CFTR rather than interference with the folding of NBD1. Interestingly, increases in the stability of NBD1 constructs are observed upon introduction of second-site mutations that suppress the trafficking defect caused by the DeltaF508 mutation, suggesting that these suppressors might function indirectly by improving the folding efficiency of NBD1 in the context of the full-length protein. The human NBD1 structures also solidify the understanding of CFTR regulation by showing that its two protein segments that can be phosphorylated both adopt multiple conformations that modulate access to the ATPase active site and functional interdomain interfaces.

  12. Regulation of CARD8 expression by ANRIL and association of CARD8 single nucleotide polymorphism rs2043211 (p.C10X) with ischemic stroke.

    PubMed

    Bai, Ying; Nie, Shaofang; Jiang, Guiqing; Zhou, Yingchao; Zhou, Mengchen; Zhao, Yuanyuan; Li, Sisi; Wang, Fan; Lv, Qiulun; Huang, Yufeng; Yang, Qin; Li, Qingxian; Li, Yue; Xia, Yunlong; Liu, Ying; Liu, Jinqiu; Qian, Jin; Li, Bin; Wu, Gang; Wu, Yanxia; Wang, Binbin; Cheng, Xiang; Yang, Yanzong; Ke, Tie; Li, Hui; Ren, Xiang; Ma, Xu; Liao, Yuhua; Xu, Chengqi; Tu, Xin; Wang, Qing K

    2014-02-01

    ANRIL has long been considered as the strongest candidate gene at the 9p21 locus, robustly associated with stroke and coronary artery disease. However, the underlying molecular mechanism remains unknown. The present study works to elucidate such a mechanism. Using expression quantitative loci analysis, we identified potential genes whose expression may be influenced by genetic variation in ANRIL. To verify the identified gene(s), knockdown and overexpression of ANRIL were evaluated in human umbilical vein endothelial cells and HepG2 cells. Ischemic stroke and coronary artery disease risk were then evaluated in the gene(s) demonstrated to be mediated by ANRIL in 3 populations of Chinese Han ancestry: 2 ischemic stroke populations consisting of the Central China cohort (903 cases and 873 controls) and the Northern China cohort (816 cases and 879 controls) and 1 coronary artery disease cohort consisting of 772 patients and 873 controls. Expression quantitative loci analysis identified CARD8 among others, with knockdown of ANRIL expression decreasing CARD8 expression and overexpression of ANRIL increasing CARD8 expression. The minor T allele of a previously identified CARD8 variant (rs2043211) was found to be significantly associated with a protective effect of ischemic stroke under the recessive model in 2 independent stroke cohorts. No significant association was found between rs2043211 and coronary artery disease. CARD8 is a downstream target gene regulated by ANRIL. Single nucleotide polymorphism rs2043211 in CARD8 is significantly associated with ischemic stroke. ANRIL may increase the risk of ischemic stroke through regulation of the CARD8 pathway.

  13. Male gametophyte-specific WRKY34 transcription factor mediates cold sensitivity of mature pollen in Arabidopsis

    PubMed Central

    Zou, Changsong; Jiang, Wenbo; Yu, Diqiu

    2010-01-01

    Mature pollen is very sensitive to cold stress in chilling-sensitive plants. Plant WRKY DNA-binding transcription factors are key regulators in plant responses to abiotic and biotic stresses. Previous studies have suggested that WRKY34 (At4g26440) gene might be involved in pollen viability, although the mechanism involved is unclear. In this study, it is shown that cold treatment increased WRKY34 expression in the wild type, and promoter-GUS analysis revealed that WRKY34 expression is pollen-specific. Enhanced green fluorescent protein-tagged WRKY34 was localized in the nuclei. Pollen harbouring the wrky34 allele showed higher viability than pollen with the WRKY34 allele after cold treatment. Further functional analysis indicated that the WRKY34 transcription factor was involved in pollen development regulated by the pollen-specific MIKC* class of MADS-domain transcription factors under cold stress, and cold-insensitivity of mature wrky34 pollen might be partly attributable to the enhanced expression of transcriptional activator CBFs in the mutants. Thus, the WRKY34 transcription factor negatively mediated cold sensitivity of mature Arabidopsis pollen and might be involved in the CBF signal cascade in mature pollen. PMID:20643804

  14. VANGUARD1 encodes a pectin methylesterase that enhances pollen tube growth in the Arabidopsis style and transmitting tract.

    PubMed

    Jiang, Lixi; Yang, Shu-Lan; Xie, Li-Fen; Puah, Ching San; Zhang, Xue-Qin; Yang, Wei-Cai; Sundaresan, Venkatesan; Ye, De

    2005-02-01

    In flowering plants, penetration of the pollen tube through stigma, style, and transmitting tract is essential for delivery of sperm nuclei to the egg cells embedded deeply within female tissues. Despite its importance in plant reproduction, little is known about the underlying molecular mechanisms that regulate the navigation of the pollen tube through the stigma, style, and transmitting tract. Here, we report the identification and characterization of an Arabidopsis thaliana gene, VANGUARD1 (VGD1) that encodes a pectin methylesterase (PME)-homologous protein of 595 amino acids and is required for enhancing the growth of pollen tubes in the style and transmitting tract tissues. VGD1 was expressed specifically in pollen grain and the pollen tube. The VGD1 protein was distributed throughout the pollen grain and pollen tube, including the plasma membrane and cell wall. Functional interruption of VGD1 reduced PME activity in the pollen to 82% of the wild type and greatly retarded the growth of the pollen tube in the style and transmitting tract, resulting in a significant reduction of male fertility. In addition, the vgd1 pollen tubes were unstable and burst more frequently when germinated and grown on in vitro culture medium, compared with wild-type pollen tubes. Our study suggests that the VGD1 product is required for growth of the pollen tube, possibly via modifying the cell wall and enhancing the interaction of the pollen tube with the female style and transmitting tract tissues.

  15. Stingless bees (Melipona subnitida) adjust brood production rather than foraging activity in response to changes in pollen stores.

    PubMed

    Maia-Silva, Camila; Hrncir, Michael; Imperatriz-Fonseca, Vera Lucia; Schorkopf, Dirk Louis P

    2016-10-01

    Highly eusocial bees (honey bees and stingless bees) sustain their colonies through periods of resource scarcity by food stored within the nest. The protein supply necessary for successful brood production is ensured through adjustments of the colonies' pollen foraging according to the availability of this resource in the environment. In honey bees Apis mellifera, in addition, pollen foraging is regulated through the broods' demand for this resource. Here, we investigated the influence of the colony's pollen store level on pollen foraging and brood production in stingless bees (Melipona subnitida). When pollen was added to the nests, colonies increased their brood production and reduced their pollen foraging within 24 h. On the other hand, when pollen reserves were removed, colonies significantly reduced their brood production. In strong contrast to A. mellifera; however, M. subnitida did not significantly increase its pollen foraging activity under poor pollen store conditions. This difference concerning the regulation of pollen foraging may be due to differences regarding the mechanism of brood provisioning. Honey bees progressively feed young larvae and, consequently, require a constant pollen supply. Stingless bees, by contrast, mass-provision their brood cells and temporary absence of pollen storage will not immediately result in substantial brood loss.

  16. Water status and associated processes mark critical stages in pollen development and functioning

    PubMed Central

    Firon, Nurit; Nepi, Massimo; Pacini, Ettore

    2012-01-01

    Background The male gametophyte developmental programme can be divided into five phases which differ in relation to the environment and pollen hydration state: (1) pollen develops inside the anther immersed in locular fluid, which conveys substances from the mother plant – the microsporogenesis phase; (2) locular fluid disappears by reabsorption and/or evaporation before the anther opens and the maturing pollen grains undergo dehydration – the dehydration phase; (3) the anther opens and pollen may be dispersed immediately, or be held by, for example, pollenkitt (as occurs in almost all entomophilous species) for later dispersion – the presentation phase; (4) pollen is dispersed by different agents, remaining exposed to the environment for different periods – the dispersal phase; and (5) pollen lands on a stigma and, in the case of a compatible stigma and suitable conditions, undergoes rehydration and starts germination – the pollen–stigma interaction phase. Scope This review highlights the issue of pollen water status and indicates the various mechanisms used by pollen grains during their five developmental phases to adjust to changes in water content and maintain internal stability. Conclusions Pollen water status is co-ordinated through structural, physiological and molecular mechanisms. The structural components participating in regulation of the pollen water level, during both dehydration and rehydration, include the exine (the outer wall of the pollen grain) and the vacuole. Recent data suggest the involvement of water channels in pollen water transport and the existence of several molecular mechanisms for pollen osmoregulation and to protect cellular components (proteins and membranes) under water stress. It is suggested that pollen grains will use these mechanisms, which have a developmental role, to cope with environmental stress conditions. PMID:22523424

  17. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbing mode.

    PubMed

    Gui, Cai-Ping; Dong, Xin; Liu, Hai-Kuan; Huang, Wei-Jie; Zhang, Dong; Wang, Shu-Jie; Barberini, María Laura; Gao, Xiao-Yan; Muschietti, Jorge; McCormick, Sheila; Tang, Wei-Hua

    2014-09-01

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane-localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing "blebs." Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca(2+)-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells. © 2014 American Society of Plant Biologists. All rights reserved.

  18. Down-regulation of P2U-purinergic nucleotide receptor messenger RNA expression during in vitro differentiation of human myeloid leukocytes by phorbol esters or inflammatory activators.

    PubMed

    Martin, K A; Kertesy, S B; Dubyak, G R

    1997-01-01

    HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5-10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was approximately 60 min, and this was not affected by acute exposure (< or = 4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by gamma-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.

  19. Regulatory Networks in Pollen Development under Cold Stress

    PubMed Central

    Sharma, Kamal D.; Nayyar, Harsh

    2016-01-01

    Cold stress modifies anthers’ metabolic pathways to induce pollen sterility. Cold-tolerant plants, unlike the susceptible ones, produce high proportion of viable pollen. Anthers in susceptible plants, when exposed to cold stress, increase abscisic acid (ABA) metabolism and reduce ABA catabolism. Increased ABA negatively regulates expression of tapetum cell wall bound invertase and monosaccharide transport genes resulting in distorted carbohydrate pool in anther. Cold-stress also reduces endogenous levels of the bioactive gibberellins (GAs), GA4 and GA7, in susceptible anthers by repression of the GA biosynthesis genes. Here, we discuss recent findings on mechanisms of cold susceptibility in anthers which determine pollen sterility. We also discuss differences in regulatory pathways between cold-stressed anthers of susceptible and tolerant plants that decide pollen sterility or viability. PMID:27066044

  20. Grass Pollen Allergens

    PubMed Central

    Augustin, Rosa

    1959-01-01

    Heat and pH stability studies and experiments with organic solvents show that the A-antigens discussed in the preceding paper (Augustin, 1959c) are much more labile than the I- (`inner ring') antigens. Breakdown products and/or aggregates are produced which no longer precipitate with antisera to the original extracts, but act as inhibitors. Solutions of pollen allergens, on the other hand, are found to withstand even autoclaving for 15 min. at 20 atm. and vigorous boiling over the naked flame of a bunsen burner. None of the carbohydrates tested has a demonstrable effect on skin reactivity which is, however, destroyed by crystalline pepsin, crystalline trypsin, a crystalline mould protease and a tissue protease (a partially purified extract from rabbit spleen). It follows that the bulk of the allergens—if not all—are proteins. The relation of skin reactivity, immuno-electrophoretic patterns, carbohydrate and protein reactions to the selective destruction of the pollen antigens is investigated. Pollen components prove to have a somewhat wider range of electrophoretic mobilities than serum proteins and are probably as complicated a mixture. The most and least highly negatively charged components are without skin reactivity in allergic subjects. The skin reactive allergens appear to have the mobilities of α- and β-globulins. Not all the hay fever subjects react equally to all the components, and Cocksfoot and Timothy activity patterns vary in different subjects. ImagesFIG. 5 PMID:13795119

  1. Mutations in Two Putative Phosphorylation Motifs in the Tomato Pollen Receptor Kinase LePRK2 Show Antagonistic Effects on Pollen Tube Length*

    PubMed Central

    Salem, Tamara; Mazzella, Agustina; Barberini, María Laura; Wengier, Diego; Motillo, Viviana; Parisi, Gustavo; Muschietti, Jorge

    2011-01-01

    The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser277/Ser279/Ser282, resulted in longer pollen tubes, but alanine substitutions in motif II, Ser304/Ser307/Thr308, resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status. PMID:21131355

  2. [Allergy, pollen and the environment].

    PubMed

    Terán, Luis Manuel; Haselbarth-López, Michelle Marie Margarete; Quiroz-García, David Leonor

    2009-01-01

    Allergic respiratory diseases such asthma and allergic rhinitis are a health problem throughout the world. In Mexico City, pollens are an important cause of allergic respiratory disease. Both, the geographic location- and the vegetation surrounding this City favor the distribution of pollens leading to respiratory disease in susceptible patients. Aerobiological studies have shown that during the mild dry winter there is a large amount of pollens in the environment with tree pollens being the most abundant of all. The most frequent tree pollens found in Mexico City include Fraxinus, Cupressaseae, Alnus, Liquidambar, Callistemon, Pinus, and Casuarina. In contrast, grass- and weed pollens predominate during the summer (rainy season) including Compositae, Cheno-Am, Ambrosia and Gramineae. An additional health problem in Mexico City is the air pollution that exerts a direct effect on individuals. This in turn increases pollen allergenicity by disrupting them leading to the release of their particles which then penetrate the human airways causing disease. Thus, the polluted environment along with global warming which is also known to increase pollen quantities by inducing longer pollen seasons may represent a health risk to Mexico City inhabitants.

  3. Age-dependent down-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 4 causes deterioration of canine sinoatrial node function.

    PubMed

    Du, Jianlin; Deng, Songbai; Pu, Di; Liu, Yajie; Xiao, Jun; She, Qiang

    2017-03-23

    The activity of pacemaker cells in the sinoatrial node (SAN) is an indicator of normal sinus rhythm. Clinical studies have revealed that the dysfunction of the SAN progressively increases with aging. In this study, we determined the changes in hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) expression and the relationship between aging and canine SAN dysfunction. The results of cardiac electrophysiological determination revealed that the intrinsic heart rate decreased from 168 ± 11 beats min-1 in young canines to 120 ± 9 beats min-1 in adults and to 88 ± 9 beats min-1 in aged canines. The sinus node recovery time (SNRT) increased from 412 ± 32 ms in young canines to 620 ± 56 ms in adults and to 838 ± 120 ms in aged canines. Corrected SNRT (CSNRT) increased from 55 ± 12 ms in young canines to 117 ± 27 ms in adults and to 171 ± 37 ms in aged canines. These results indicated that SAN function deteriorated with aging in the canine heart. However, histological staining illustrated that fibrosis was not significantly increased with aging in canine SAN. Real-time polymerase chain reaction indicated that the expression of HCN4 mRNA was downregulated in the elderly canine SAN. Similarly, we also verified that HCN4 protein expression within the SAN declined with aging via immunofluorescence staining and western blot analysis. Taken together, our data show that electrical remodeling, related to the down-regulation of HCN4, is responsible for the gradually increased incidence of SAN dysfunction with aging. Our results provide further evidence for explaining the mechanisms of age-related deterioration in the SAN.

  4. Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain.

    PubMed

    Shah, Rachit; Chou, Tsui-Fen; Maize, Kimberly M; Strom, Alexander; Finzel, Barry C; Wagner, Carston R

    2017-09-23

    Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd(2+), Cu(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+)) on the structural integrity and catalytic activity of hHint1. With the exception of Mg(2+), all the divalent ions inhibited hHint1, the rank of order was found to be Cu(2+) >Zn(2+) >Cd(2+) ≥Ni(2+) >Mn(2+) based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu(2+) is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu(2+); restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Deletion of Phenylalanine 508 in the First Nucleotide-binding Domain of the Cystic Fibrosis Transmembrane Conductance Regulator Increases Conformational Exchange and Inhibits Dimerization.

    PubMed

    Chong, P Andrew; Farber, Patrick J; Vernon, Robert M; Hudson, Rhea P; Mittermaier, Anthony K; Forman-Kay, Julie D

    2015-09-18

    Deletion of Phe-508 (F508del) in the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) results in destabilization of the domain, intramolecular interactions involving the domain, and the entire channel. The destabilization caused by F508del manifests itself in defective channel processing and channel gating defects. Here, we present NMR studies of the effect of F508del and the I539T stabilizing mutation on NBD1 dynamics, with a view to understanding these changes in stability. Qualitatively, F508del NMR spectra exhibit significantly more peak broadening than WT spectra due to the enhanced intermediate time scale (millisecond to microsecond) motions in the mutant. Unexpectedly, studies of fast (nanosecond to picosecond) motions revealed that F508del NBD1 tumbles more rapidly in solution than WT NBD1. Whereas F508del tumbles at a rate nearly consistent with the monomeric state, the WT protein tumbles significantly more slowly. Paramagnetic relaxation enhancement experiments confirm that NBD1 homodimerizes in solution in the expected head-to-tail orientation. NMR spectra of WT NBD1 reveal significant concentration-dependent chemical shift perturbations consistent with NBD1 dimerization. Chemical shift analysis suggests that the more rapid tumbling of F508del is the result of an impaired ability to dimerize. Based on previously published crystal structures and NMR spectra of various NBD1 mutants, we propose that deletion of Phe-508 affects Q-loop conformational sampling in a manner that inhibits dimerization. These results provide a potential mechanism for inhibition of channel opening by F508del and support the dimer interface as a target for cystic fibrosis therapeutics.

  6. The B3 Subunit of the Cone Cyclic Nucleotide-gated Channel Regulates the Light Responses of Cones and Contributes to the Channel Structural Flexibility*

    PubMed Central

    Ding, Xi-Qin; Thapa, Arjun; Ma, Hongwei; Xu, Jianhua; Elliott, Michael H.; Rodgers, Karla K.; Smith, Marci L.; Wang, Jin-Shan; Pittler, Steven J.; Kefalov, Vladimir J.

    2016-01-01

    Cone photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in cone phototransduction, which is a process essential for daylight vision, color vision, and visual acuity. Mutations in the cone channel subunits CNGA3 and CNGB3 are associated with human cone diseases, including achromatopsia, cone dystrophies, and early onset macular degeneration. Mutations in CNGB3 alone account for 50% of reported cases of achromatopsia. This work investigated the role of CNGB3 in cone light response and cone channel structural stability. As cones comprise only 2–3% of the total photoreceptor population in the wild-type mouse retina, we used Cngb3−/−/Nrl−/− mice with CNGB3 deficiency on a cone-dominant background in our study. We found that, in the absence of CNGB3, CNGA3 was able to travel to the outer segments, co-localize with cone opsin, and form tetrameric complexes. Electroretinogram analyses revealed reduced cone light response amplitude/sensitivity and slower response recovery in Cngb3−/−/Nrl−/− mice compared with Nrl−/− mice. Absence of CNGB3 expression altered the adaptation capacity of cones and severely compromised function in bright light. Biochemical analysis demonstrated that CNGA3 channels lacking CNGB3 were more resilient to proteolysis than CNGA3/CNGB3 channels, suggesting a hindered structural flexibility. Thus, CNGB3 regulates cone light response kinetics and the channel structural flexibility. This work advances our understanding of the biochemical and functional role of CNGB3 in cone photoreceptors. PMID:26893377

  7. Genetic manipulation of a transcription-regulating sequence of porcine reproductive and respiratory syndrome virus reveals key nucleotides determining its activity.

    PubMed

    Zheng, Haihong; Zhang, Keyu; Zhu, Xing-Quan; Liu, Changlong; Lu, Jiaqi; Gao, Fei; Zhou, Yan; Zheng, Hao; Lin, Tao; Li, Liwei; Tong, Guangzhi; Wei, Zuzhang; Yuan, Shishan

    2014-08-01

    The factors that determine the transcription-regulating sequence (TRS) activity of porcine reproductive and respiratory syndrome virus (PRRSV) remain largely unclear. In this study, the effect of mutagenesis of conserved C nucleotides at positions 5 and 6 in the leader TRS (TRS-L) and/or canonical body TRS7 (TRS-B7) on the synthesis of subgenomic (sg) mRNA and virus infectivity was investigated in the context of a type 2 PRRSV infectious cDNA clone. The results showed that a double C mutation in the leader TRS completely abolished sg mRNAs synthesis and virus infectivity, but a single C mutation did not. A single C or double C mutation in TRS-B7.1 or/and TRS-B7.2 impaired or abolished the corresponding sg mRNA synthesis. Introduction of identical mutations in the leader and body TRSs partially restored sg mRNA7.1 and/or sg mRNA7.2 transcription, indicating that the base-pairing interaction between sense TRS-L and cTRS-B is a crucial factor influencing sg mRNA synthesis. Analysis of the mRNA leader-body junctions of mutants provided evidence for a mechanism of discontinuous minus-strand transcription. This study also showed that mutational inactivation of TRS-B7.1 or TRS-B7.2 did not affect the production of infectious progeny virus, and the sg mRNA formed from each of them could express N protein. However, TRS-B7.1 plays more important roles than TRS-B7.2 in maintaining the growth characteristic of type 2 PRRSV. These results provide more insight into the molecular mechanism of genome expression and subgenomic mRNA transcription of PRRSV.

  8. An ankyrin repeat-containing protein, characterized as a ubiquitin ligase, is closely associated with membrane-enclosed organelles and required for pollen germination and pollen tube growth in lily.

    PubMed

    Huang, Jian; Chen, Feng; Del Casino, Cecilia; Autino, Antonella; Shen, Mouhua; Yuan, Shuai; Peng, Jia; Shi, Hexin; Wang, Chen; Cresti, Mauro; Li, Yiqin

    2006-04-01

    Exhibiting rapid polarized growth, the pollen tube delivers the male gametes into the ovule for fertilization in higher plants. To get an overall picture of gene expression during pollen germination and pollen tube growth, we profiled the transcription patterns of 1,536 pollen cDNAs from lily (Lilium longiflorum) by microarray. Among those that exhibited significant differential expression, a cDNA named lily ankyrin repeat-containing protein (LlANK) was thoroughly studied. The full-length LlANK cDNA sequence predicts a protein containing five tandem ankyrin repeats and a RING zinc-finger domain. The LlANK protein possesses ubiquitin ligase activity in vitro. RNA blots demonstrated that LlANK transcript is present in mature pollen and its level, interestingly contrary to most pollen mRNAs, up-regulated significantly during pollen germination and pollen tube growth. When fused with green fluorescent protein and transiently expressed in pollen, LlANK was found dominantly associated with membrane-enclosed organelles as well as the generative cell. Overexpression of LlANK, however, led to abnormal growth of the pollen tube. On the other hand, transient silencing of LlANK impaired pollen germination and tube growth. Taken together, these results showed that LlANK is a ubiquitin ligase associated with membrane-enclosed organelles and required for polarized pollen tube growth.

  9. Pollen-Specific Aquaporins NIP4;1 and NIP4;2 Are Required for Pollen Development and Pollination in Arabidopsis thaliana.

    PubMed

    Di Giorgio, Juliana Andrea Pérez; Bienert, Gerd Patrick; Ayub, Nicolás Daniel; Yaneff, Agustín; Barberini, María Laura; Mecchia, Martín Alejandro; Amodeo, Gabriela; Soto, Gabriela Cynthia; Muschietti, Jorge Prometeo

    2016-05-01

    In flowers with dry stigmas, pollen development, pollination, and pollen tube growth require spatial and temporal regulation of water and nutrient transport. To better understand the molecular mechanisms involved in reproductive processes, we characterized NIP4;1 and NIP4;2, two pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 are paralogs found exclusively in the angiosperm lineage. Although they have 84% amino acid identity, they displayed different expression patterns. NIP4;1 has low expression levels in mature pollen, while NIP4;2 expression peaks during pollen tube growth. Additionally, NIP4;1pro:GUS flowers showed GUS activity in mature pollen and pollen tubes, whereas NIP4;2pro:GUS flowers only in pollen tubes. Single T-DNA mutants and double artificial microRNA knockdowns had fewer seeds per silique and reduced pollen germination and pollen tube length. Transport assays in oocytes showed NIP4;1 and NIP4;2 function as water and nonionic channels. We also found that NIP4;1 and NIP4;2 C termini are phosphorylated by a pollen-specific CPK that modifies their water permeability. Survival assays in yeast indicated that NIP4;1 also transports ammonia, urea, boric acid, and H2O2 Thus, we propose that aquaporins NIP4;1 and NIP4;2 are exclusive components of the reproductive apparatus of angiosperms with partially redundant roles in pollen development and pollination.

  10. Pollen Tube Growth Regulation by Free Anions Depends on the Interaction between the Anion Channel SLAH3 and Calcium-Dependent Protein Kinases CPK2 and CPK20[C][W

    PubMed Central

    Gutermuth, Timo; Lassig, Roman; Portes, Maria-Teresa; Maierhofer, Tobias; Romeis, Tina; Borst, Jan-Willem; Hedrich, Rainer; Feijó, José A.; Konrad, Kai R.

    2013-01-01

    Apical growth in pollen tubes (PTs) is associated with the presence of tip-focused ion gradients and fluxes, implying polar localization or regulation of the underlying transporters. The molecular identity and regulation of anion transporters in PTs is unknown. Here we report a negative gradient of cytosolic anion concentration focused on the tip, in negative correlation with the cytosolic Ca2+ concentration. We hypothesized that a possible link between these two ions is based on the presence of Ca2+-dependent protein kinases (CPKs). We characterized anion channels and CPK transcripts in PTs and analyzed their localization. Yellow fluorescent protein (YFP) tagging of a homolog of SLOW ANION CHANNEL-ASSOCIATED1 (SLAH3:YFP) was widespread along PTs, but, in accordance with the anion efflux, CPK2/CPK20/CPK17/CPK34:YFP fluorescence was strictly localized at the tip plasma membrane. Expression of SLAH3 with either CPK2 or CPK20 (but not CPK17/CPK34) in Xenopus laevis oocytes elicited S-type anion channel currents. Interaction of SLAH3 with CPK2/CPK20 (but not CPK17/CPK34) was confirmed by Förster-resonance energy transfer fluorescence lifetime microscopy in Arabidopsis thaliana mesophyll protoplasts and bimolecular fluorescence complementation in living PTs. Compared with wild-type PTs, slah3-1 and slah3-2 as well as cpk2-1 cpk20-2 PTs had reduced anion currents. Double mutant cpk2-1 cpk20-2 and slah3-1 PTs had reduced extracellular anion fluxes at the tip. Our studies provide evidence for a Ca2+-dependent CPK2/CPK20 regulation of the anion channel SLAH3 to regulate PT growth. PMID:24280384

  11. Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis*

    PubMed Central

    He, Jinshan; Zhu, Qianzheng; Wani, Gulzar; Sharma, Nidhi; Han, Chunhua; Qian, Jiang; Pentz, Kyle; Wang, Qi-en; Wani, Altaf A.

    2014-01-01

    Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis. PMID:25118285

  12. Antisense Oligodeoxynucleotide Inhibition as an Alternative and Convenient Method for Gene Function Analysis in Pollen Tubes

    PubMed Central

    Liao, Fanglei; Wang, Lu; Yang, Li-Bo; Zhang, Liyao; Peng, Xiongbo; Sun, Meng-xiang

    2013-01-01

    Antisense oligodeoxynucleotide (A-ODN) inhibition works well in animal cells. However, there have been few successful examples to date of its application in plants, and more specifically whether the technique can be used in pollen tubes as a model of plant cell growth. NtGNL1 plays an important role in pollen tube development and was thus selected as an indicator to assess the biological effects of A-ODN. An A-ODN inhibition technique was used to down-regulate NtGNL1 expression in tobacco pollen tubes and showed that A-ODNs could quickly enter pollen tubes through the thick wall and cell membrane and effectively block NtGNL1 expression. Phenotype analysis revealed that the down-regulation of NtGNL1 by A-ODNs resulted in abnormalities in endocytosis and subsequent vesicle trafficking, similar to the phenotypes of pollen tubes treated with NtGNL1 RNAi. This investigation confirmed that A-ODNs could specifically inhibit target gene expression, and furthermore demonstrated that A-ODN functioned in a concentration- and duration-dependent manner, because A-ODNs could be degraded when incubated with pollen tubes. Thus, the A-ODN technique was successfully used for gene function analysis in pollen tubes and appears to be an alternative and convenient technique when the in vitro pollen tube is used as the study model. This technique will greatly facilitate investigations on the molecular mechanism(s) underlying pollen tube growth. PMID:23527102

  13. [Cypress pollen allergy].

    PubMed

    Charpin, D; Calleja, M; Pichot, C; Penel, V; Hugues, B; Poncet, P

    2013-12-01

    Cypress belongs to the Cupressaceae family, which includes 140 species with non-deciduous foliage. The most important genera in allergic diseases are Cupressus sempervirens or Green cypress, Cupressus arizonica or Blue cypress, Juniperus oxycedrus, Juniperus communis and Thuya. Because J. oxycedrus pollinates in October, C. sempervirens in January and February, C. arizonica in February and March, J. communis in April, the symptomatic period is long-lasting. Because of global warming, the pollination period is tending to last longer and Cupressaceae species are becoming established further the north. In Mediterranean countries, cypress is by far the most important pollinating species, accounting for half of the total pollination. The major allergens belong to group 1. The other allergens from cypress and Juniper share 75 to 97 % structural homology with group 1 major allergens. The prevalence of cypress allergy in the general population ranges from 5 % to 13 %, according to exposure to the pollen. Among outpatients consulting an allergist, between 9 and 35 %, according to different studies, are sensitized to cypress pollen. Repeated cross-sectional studies performed at different time intervals have demonstrated a threefold increase in the percentage of cypress allergy. Risk factors include a genetic predisposition and/or a strong exposure to pollen, but air pollutants could play a synergistic role. The study of the natural history of cypress allergy allows the identification of a subgroup of patients who have no personal or family history of atopy, whose disease began later in life, with low total IgE and often monosensitization to cypress pollen. In these patients, the disease is allergic than rather atopic. In the clinical picture, rhinitis is the most prevalent symptom but conjunctivitis the most disabling. A cross-reactivity between cypress and peach allergy has been demonstrated. The pharmacological treatment of cypress allergy is not different from

  14. Contribution of pollen to atmospheric ice nuclei concentrations

    NASA Astrophysics Data System (ADS)

    Hader, J. D.; Wright, T. P.; Petters, M. D.

    2013-12-01

    Recent studies have suggested that the ice nucleating ability of some types of pollen is derived from non-proteinaceous macromolecules. These macromolecules may become dispersed by the rupturing of the pollen sac during wetting and drying cycles in the atmosphere. If true, this mechanism might prove to be a significant source of ice nuclei (IN) concentrations when pollen are present. Here we test this hypothesis by measuring ambient IN concentrations from the beginning to the end of the 2013 pollen season in Raleigh, North Carolina, USA. Air samples were collected using a swirling aerosol collector twice per week and the solutions were analysed for ice nuclei activity using a droplet freezing assay. Rainwater samples were collected at the peak of the pollen season and analysed with the drop freezing assay to compare the potentially enhanced IN concentrations measured near the ground with IN concentrations found aloft. Ambient ice nuclei spectra, defined as the number of ice nuclei per volume of air as a function of temperature, are inferred from the aerosol collector solutions. No general trend was observed between ambient pollen counts and observed IN concentrations, suggesting that ice nuclei multiplication via pollen sac rupturing and subsequent release of macromolecules was not prevalent for the pollen types and meteorological conditions typically encountered in the Southeastern US. A serendipitously sampled collection after a downpour provided evidence for a rain-induced IN burst with an observed IN concentration of approximately 30 per litre, a 30-fold increase over background concentrations at -20 °C. The onset temperature of freezing for these particles was approximately -12 °C, suggesting that the ice nucleating particles were biological in origin. The magnitude of the IN burst was significantly larger than previously observed, providing additional evidence to merit further investigation of a self-regulated feedback cycle between the atmosphere and

  15. Interference of the Histone Deacetylase Inhibits Pollen Germination and Pollen Tube Growth in Picea wilsonii Mast

    PubMed Central

    Zhou, Junhui; Li, Xiaojuan

    2015-01-01

    Histone deacetylase (HDAC) is a crucial component in the regulation of gene expression in various cellular processes in animal and plant cells. HDAC has been reported to play a role in embryogenesis. However, the effect of HDAC on androgamete development remains unclear, especially in gymnosperms. In this study, we used the HDAC inhibitors trichostatin A (TSA) and sodium butyrate (NaB) to examine the role of HDAC in Picea wilsonii pollen germination and pollen tube elongation. Measurements of the tip-focused Ca2+ gradient revealed that TSA and NaB influenced this gradient. Immunofluorescence showed that actin filaments were disrupted into disorganized fragments. As a result, the vesicle trafficking was disturbed, as determined by FM4-64 labeling. Moreover, the distribution of pectins and callose in cell walls was significantly altered in response to TSA and NaB. Our results suggest that HDAC affects pollen germination and polarized pollen tube growth in Picea wilsonii by affecting the intracellular Ca2+ concentration gradient, actin organization patterns, vesicle trafficking, as well as the deposition and configuration of cell wall components. PMID:26710276

  16. IRREGULAR POLLEN EXINE1 Is a Novel Factor in Anther Cuticle and Pollen Exine Formation1[OPEN

    PubMed Central

    Chen, Xiaoyang; Zhang, Hua; Luo, Hongbing; Zhao, Li; Dong, Zhaobin; Yan, Shuangshuang; Liu, Renyi; Xu, Chunyan; Li, Song; Chen, Huabang

    2017-01-01

    Anther cuticle and pollen exine are protective barriers for pollen development and fertilization. Despite that several regulators have been identified for anther cuticle and pollen exine development in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), few genes have been characterized in maize (Zea mays) and the underlying regulatory mechanism remains elusive. Here, we report a novel male-sterile mutant in maize, irregular pollen exine1 (ipe1), which exhibited a glossy outer anther surface, abnormal Ubisch bodies, and defective pollen exine. Using map-based cloning, the IPE1 gene was isolated as a putative glucose-methanol-choline oxidoreductase targeted to the endoplasmic reticulum. Transcripts of IPE1 were preferentially accumulated in the tapetum during the tetrad and early uninucleate microspore stage. A biochemical assay indicated that ipe1 anthers had altered constituents of wax and a significant reduction of cutin monomers and fatty acids. RNA sequencing data revealed that genes implicated in wax and flavonoid metabolism, fatty acid synthesis, and elongation were differentially expressed in ipe1 mutant anthers. In addition, the analysis of transfer DNA insertional lines of the orthologous gene in Arabidopsis suggested that IPE1 and their orthologs have a partially conserved function in male organ development. Our results showed that IPE1 participates in the putative oxidative pathway of C16/C18 ω-hydroxy fatty acids and controls anther cuticle and pollen exine development together with MALE STERILITY26 and MALE STERILITY45 in maize. PMID:28049856

  17. Grass Pollen Allergens

    PubMed Central

    Augustin, Rosa

    1959-01-01

    Grass pollen allergens are shown to remain associated with protein material and a yellow pigment during paper chromatography and during dialyses and ultrafiltrations of various types. Dialysable* allergens comprise only a fraction of 1 per cent of the total activity and the amount of activity extractable by diethylene glycol (DEG) and similar solvents is of the same order. Besides the allergens, the DEG and aqueous extracts contain large amounts of inositol, glucose and fructose, also some yellow pigments and phosphates. Larger amounts of free and combined amino acids are found in the aqueous than in the DEG extracts, but the reverse is true for sucrose. In addition the DEG extracts contain a yellow glucoside different from the dactylen of the aqueous extracts, a glucosan and an arabinose-galactose-pigment complex, only the latter being associated with any activity. The spontaneous release of the crystalline dactylen from originally clear aqueous pollen extracts is found not to be caused by enzymes. The washed crystals are found to be chromatographically and electrophoretically homogeneous and devoid of allergenic activity. ImagesFIG. 1FIG. 2FIG. 3FIG. 4FIG. 5FIG. 6FIG. 7 PMID:13640676

  18. A Simple Method for Collecting Airborne Pollen

    ERIC Educational Resources Information Center

    Kevan, Peter G.; DiGiovanni, Franco; Ho, Rong H.; Taki, Hisatomo; Ferguson, Kristyn A.; Pawlowski, Agata K.

    2006-01-01

    Pollination is a broad area of study within biology. For many plants, pollen carried by wind is required for successful seed set. Airborne pollen also affects human health. To foster studies of airborne pollen, we introduce a simple device--the "megastigma"--for collecting pollen from the air. This device is flexible, yielding easily obtained data…

  19. A Simple Method for Collecting Airborne Pollen

    ERIC Educational Resources Information Center

    Kevan, Peter G.; DiGiovanni, Franco; Ho, Rong H.; Taki, Hisatomo; Ferguson, Kristyn A.; Pawlowski, Agata K.

    2006-01-01

    Pollination is a broad area of study within biology. For many plants, pollen carried by wind is required for successful seed set. Airborne pollen also affects human health. To foster studies of airborne pollen, we introduce a simple device--the "megastigma"--for collecting pollen from the air. This device is flexible, yielding easily obtained data…

  20. CHARACTERIZATION OF THE MAIZE POLLEN TRANSCRIPTOME

    EPA Science Inventory

    Pollen is a primary vehicle for transgene flow from engineered plants to their non-transgenic, native or weedy relatives. Hence, gene flow will be affected by pollen fitness (e.g., how well a particular pollen grain can outcompete other pollen present on the stigma and complete ...

  1. CHARACTERIZATION OF THE MAIZE POLLEN TRANSCRIPTOME

    EPA Science Inventory

    Pollen is a primary vehicle for transgene flow from engineered plants to their non-transgenic, native or weedy relatives. Hence, gene flow will be affected by pollen fitness (e.g., how well a particular pollen grain can outcompete other pollen present on the stigma and complete ...

  2. Pollen tube access to the ovule is mediated by glycoprotein secretion on the obturator of apple (Malus × domestica, Borkh).

    PubMed

    Losada, Juan M; Herrero, Maria

    2017-04-01

    Within the ovary, the obturator bridges the pathway of the pollen tube from the style to the ovule. Despite its widespread presence among flowering plants, its function has only been studied in a handful of species, and the molecules involved in pollen tube-obturator cross-talk have not been explored hitherto. This work evaluates the involvement of glucans and glycoproteins on pollen tube growth in the obturator of apple flowers ( Malus × domestica) . Pollen tube kinetics were sequentially examined in the pistil and related to changes occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate between changes in the obturator induced by pollen tubes from those developmentally regulated, both pollinated and unpollinated pistils were examined. Pollen tube growth rates were slow in the stigma, faster in the style and slow again in the ovary. The arrival of pollen tubes at the obturator was concomitant with the secretion of proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan proteins (AGPs). While some of these secretions - extensins and AGPs labelled by JIM13 - were developmentally regulated, others - AGPs labelled by JIM8 - were elicited by the presence of pollen tubes. Following pollen tube passage, all these glycoproteins were depleted. The results show a timely secretion of glycoproteins on the obturator surface concomitant with pollen tube arrival at this structure. The fact that their secretion is depleted following pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. Remarkably, both the regulation of the secretion of the different glycoproteins, as well as their association with the performance of pollen tubes exhibit similarities with those observed in the stigma, in line with their common developmental origin.

  3. City scale pollen concentration variability

    NASA Astrophysics Data System (ADS)

    van der Molen, Michiel; van Vliet, Arnold; Krol, Maarten

    2016-04-01

    Pollen are emitted in the atmosphere both in the country-side and in cities. Yet the majority of the population is exposed to pollen in cities. Allergic reactions may be induced by short-term exposure to pollen. This raises the question how variable pollen concentration in cities are in temporally and spatially, and how much of the pollen in cities are actually produced in the urban region itself. We built a high resolution (1 × 1 km) pollen dispersion model based on WRF-Chem to study a city's pollen budget and the spatial and temporal variability in concentration. It shows that the concentrations are highly variable, as a result of source distribution, wind direction and boundary layer mixing, as well as the release rate as a function of temperature, turbulence intensity and humidity. Hay Fever Forecasts based on such high resolution emission and physical dispersion modelling surpass traditional hay fever warning methods based on temperature sum methods. The model gives new insights in concentration variability, personal and community level exposure and prevention. The model will be developped into a new forecast tool to serve allergic people to minimize their exposure and reduce nuisance, coast of medication and sick leave. This is an innovative approach in hay fever warning systems.

  4. [Japanese cedar pollen in house dust].

    PubMed

    Enomoto, T; Ohnishi, S; Dake, Y; Sakoda, T; Saitoh, Y; Sogo, H; Seno, S; Fujiki, Y; Fujimura, S

    2001-06-01

    Caring for oneself against Japanese cedar pollinosis is important as well as receiving medical-care. Although the importance of avoiding pollen is described in the guideline for nasal allergy medical treatment, however, there is no information for effective dust cleaning for the home. This study examined how many cedar pollens were included in indoor dust in order to obtain basic data whether dust removal for cedar pollen is available for pollinosis suffers. As a result, the study found that there were many Japanese cedar pollens in indoor dust even before the pollen season. Cedar pollen increased with the increasing number of airborne pollen. The highest number of pollen found in one week was approximately 450 pollens in a square meter of a living room floor. The study concluded that cleaning is one of the best way to remove Japanese cedar pollens found in indoor dust.

  5. Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides.

    PubMed

    Zhang, Xiao-Ling; Zhang, Jin; Guo, Ying-Hua; Sun, Pei; Jia, Hui-Xia; Fan, Wei; Lu, Meng-Zhu; Hu, Jian-Jun

    2016-09-03

    Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar 'ZhongHuai1' ('ZH1', triploid) and 'ZhongHuai2' ('ZH2', diploid) generated from the same parents. The mature pollen was sterile in triploid poplar 'ZH1'. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with "S-adenosylmethionine metabolism", "actin cytoskeleton organization", or "translational elongation". The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties ('ZH1', 'ZH2', and '2KEN8'). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity.

  6. Arabidopsis microtubule-destabilizing protein 25 functions in pollen tube growth by severing actin filaments.

    PubMed

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament-severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca(2+), in vitro. Analysis of a mutant that bears a point mutation at the Ca(2+) binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca(2+) level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament-severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth.

  7. Does insect netting affect the containment of airborne pollen from (GM-) plants in greenhouses?

    PubMed

    van Hengstum, Thomas; Hooftman, Danny A P; den Nijs, Hans C M; van Tienderen, Peter H

    2012-09-01

    Greenhouses are a well-accepted containment strategy to grow and study genetically modified plants (GM) before release into the environment. Various containment levels are requested by national regulations to minimize GM pollen escape. We tested the amount of pollen escaping from a standard greenhouse, which can be used for EU containment classes 1 and 2. More specifically, we investigated the hypothesis whether pollen escape could be minimized by insect-proof netting in front of the roof windows, since the turbulent airflow around the mesh wiring could avoid pollen from escaping. We studied the pollen flow out of greenhouses with and without insect netting of two non-transgenic crops, Ryegrass (Loliummultiflorum) and Corn (Zea Mays). Pollen flow was assessed with Rotorod(®) pollen samplers positioned inside and outside the greenhouse' roof windows. A significant proportion of airborne pollen inside the greenhouse leaves through roof windows. Moreover, the lighter pollen of Lolium escaped more readily than the heavier pollen of Maize. In contrast to our expectations, we did not identify any reduction in pollen flow with insect netting in front of open windows, even under induced airflow conditions. We conclude that insect netting, often present by default in greenhouses, is not effective in preventing pollen escape from greenhouses of wind-pollinated plants for containment classes 1 or 2. Further research would be needed to investigate whether other alternative strategies, including biotic ones, are more effective. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10453-011-9237-8) contains supplementary material, which is available to authorized users.

  8. Grass Pollen Allergens

    PubMed Central

    Augustin, Rosa; Hayward, Barbara J.

    1962-01-01

    Cocksfoot and Timothy pollen extracts are each found to contain at least fifteen components antigenic in rabbits. Most of these can also be allergens for man, but only a few are regularly so. These `principal' allergens have now been isolated in highly purified form. Procedures are given for a simple method of preparing extracts for clinical purposes and for the partial separation, concentration and purification of the allergens by means of differential extractions of the pollens and by means of ultrafiltration, isoelectric precipitation and salt fractionations (at acid and neutral pH) of the extracts. Isoelectric precipitations gave highly pigmented acid complexes, two of which moved as single sharp peaks at pH 7.4 in free electrophoresis, but proved to be hardly active by skin tests. Acid NaCl fractionation of the remainder resulted for Cocksfoot and Timothy in the isolation of a nearly white powder (T21.111121112 = T21B) which was weight for weight 1000–10,000 times as active as the pollen from which it had been derived. The powders have retained their activity for 7 years. By gel diffusion tests, they were found to contain two antigens (one in each preparation) which were immunologically partially related, but the Timothy preparation contained in addition the `innermost' `twin' antigens specific for Timothy that we had discovered previously in the crude extracts by gel diffusion methods. Skin reactions could be elicited in hay-fever subjects by prick tests with concentrations of 10-9–10-8 g./ml., which is equivalent to intradermal injections of 10-11–10-10 mg. and represents a 300-fold purification with respect to the concentrates of crude pollen extracts prepared by ultrafiltration and dialysis. Fractionation on DEAE-cellulose of one of the highly purified Timothy preparations (T21.11112112 = T21A) and other, crude Timothy and Cocksfoot extracts resulted in considerable and reproducible separation of the various antigens, with no indication of the

  9. Systematically frameshifting by deletion of every 4th or 4th and 5th nucleotides during mitochondrial transcription: RNA self-hybridization regulates delRNA expression.

    PubMed

    Seligmann, Hervé

    2016-01-01

    In mitochondria, secondary structures punctuate post-transcriptional RNA processing. Recently described transcripts match the human mitogenome after systematic deletions of every 4th, respectively every 4th and 5th nucleotides, called delRNAs. Here I explore predicted stem-loop hairpin formation by delRNAs, and their associations with delRNA transcription and detected peptides matching their translation. Despite missing 25, respectively 40% of the nucleotides in the original sequence, del-transformed sequences form significantly more secondary structures than corresponding randomly shuffled sequences, indicating biological function, independently of, and in combination with, previously detected delRNA and thereof translated peptides. Self-hybridization decreases delRNA abundances, indicating downregulation. Systematic deletions of the human mitogenome reveal new, unsuspected coding and structural informations.

  10. Mammalian Mon2/Ysl2 regulates endosome-to-Golgi trafficking but possesses no guanine nucleotide exchange activity toward Arl1 GTPase

    NASA Astrophysics Data System (ADS)

    Mahajan, Divyanshu; Boh, Boon Kim; Zhou, Yan; Chen, Li; Cornvik, Tobias Carl; Hong, Wanjin; Lu, Lei

    2013-11-01

    Arl1 is a member of Arf family small GTPases that is essential for the organization and function of Golgi complex. Mon2/Ysl2, which shares significant homology with Sec7 family Arf guanine nucleotide exchange factors, was poorly characterized in mammalian cells. Here, we report the first in depth characterization of mammalian Mon2. We found that Mon2 localized to trans-Golgi network which was dependent on both its N and C termini. The depletion of Mon2 did not affect the Golgi localized or cellular active form of Arl1. Furthermore, our in vitro assay demonstrated that recombinant Mon2 did not promote guanine nucleotide exchange of Arl1. Therefore, our results suggest that Mon2 could be neither necessary nor sufficient for the guanine nucleotide exchange of Arl1. We demonstrated that Mon2 was involved in endosome-to-Golgi trafficking as its depletion accelerated the delivery of furin and CI-M6PR to Golgi after endocytosis.

  11. MYB97, MYB101 and MYB120 Function as Male Factors That Control Pollen Tube-Synergid Interaction in Arabidopsis thaliana Fertilization

    PubMed Central

    Zhu, Lei; Niu, Qian-Kun; Zhou, Jing-Jing; Li, Meng; Chen, Li-Qun; Zhang, Xue-Qin; Ye, De

    2013-01-01

    Pollen tube reception involves a pollen tube-synergid interaction that controls the discharge of sperm cells into the embryo sac during plant fertilization. Despite its importance in the sexual reproduction of plants, little is known about the role of gene regulation in this process. We report here that the pollen-expressed transcription factors MYB97, MYB101 and MYB120 probably control genes whose encoded proteins play important roles in Arabidopsis thaliana pollen tube reception. They share a high amino acid sequence identity and are expressed mainly in mature pollen grains and pollen tubes. None of the single or double mutants of these three genes exhibited any visible defective phenotype. Although the myb97 myb101 myb120 triple mutant was not defective in pollen development, pollen germination, pollen tube growth or tube guidance, the pollen tubes of the triple mutants exhibited uncontrolled growth and failed to discharge their sperm cells after entering the embryo sac. In addition, the myb97 myb101 myb120 triple mutation significantly affected the expression of a group of pollen-expressed genes in mature pollen grains. All these results indicate that MYB97, MYB101 and MYB120 participate in pollen tube reception, possibly by controlling the expression of downstream genes. PMID:24278028

  12. Pollen Aquaporins: The Solute Factor.

    PubMed

    Pérez Di Giorgio, Juliana A; Soto, Gabriela C; Muschietti, Jorge P; Amodeo, Gabriela

    2016-01-01

    In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen's success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange.

  13. Temperature-dependent effects of cadmium and purine nucleotides on mitochondrial aconitase from a marine ectotherm, Crassostrea virginica: a role of temperature in oxidative stress and allosteric enzyme regulation.

    PubMed

    Cherkasov, Anton A; Overton, Robert A; Sokolov, Eugene P; Sokolova, Inna M

    2007-01-01

    Temperature and heavy metals such as cadmium (Cd) are important environmental stressors that can strongly affect mitochondrial function of marine poikilotherms. In this study, we investigated the combined effects of temperature (20 degrees C and 30 degrees C) and Cd stress on production of reactive oxygen species (ROS) and oxidative stress in a marine poikilotherm Crassostrea virginica (the eastern oyster) using mitochondrial aconitase as a sensitive biomarker of oxidative damage. We also assessed potential involvement of mitochondrial uncoupling proteins (UCPs) in antioxidant protection in oyster mitochondria using purine nucleotides (GDP, ATP and ADP) as specific inhibitors, and free fatty acids as stimulators, of UCPs. Our results show that exposure to Cd results in elevated ROS production and oxidative damage as indicated by aconitase inactivation which is particularly pronounced at elevated temperature. Unexpectedly, oyster mitochondrial aconitase was inhibited by physiologically relevant levels of ATP (IC(50)=1.93 and 3.04 mmol l(-1) at 20 degrees C and 30 degrees C, respectively), suggesting that allosteric regulation of aconitase by this nucleotide may be involved in regulation of the tricarboxylic acid flux in oysters. Aconitase was less sensitive to ATP inhibition at 30 degrees C than at 20 degrees C, consistent with the elevated metabolic flux at higher temperatures. ADP and GDP also inhibited mitochondrial aconitase but at the levels well above the physiological concentrations of these nucleotides (6-11 mmol l(-1)). Our study shows expression of at least three UCP isoforms in C. virginica gill tissues but provides no indication that UCPs protect mitochondrial aconitase from oxidative inactivation in oysters. Overall, the results of this study indicate that temperature stress exaggerates toxicity of Cd leading to elevated oxidative stress in mitochondria, which may have important implications for survival of poikilotherms in polluted environments during

  14. Atmospheric pollen count in Monterrey, Mexico.

    PubMed

    González-Díaz, Sandra N; Rodríguez-Ortiz, Pablo G; Arias-Cruz, Alfredo; Macías-Weinmann, Alejandra; Cid-Guerrero, Dagoberto; Sedo-Mejia, Giovanni A

    2010-01-01

    There are few reports of pollen count and identification in Mexico; therefore, it is important to generate more information on the subject. This study was designed to describe the prevalence of pollen in the city of Monterrey, Mexico, during the year 2004. Atmospheric pollen was collected with a Hirst air sampler, with an airflow of 10 L/minute during 2004. Pollen was identified with light microscopy; the average monthly pollen count as well as total was calculated from January 2004 to January 2005. The months with the highest concentration of pollen were February and March (289 and 142 grains/m(3) per day, respectively), and July and November had the lowest concentration (20 and 11 grains/m(3) per day, respectively). Most of the pollen recollected corresponded to tree pollen (72%). Fraxinus spp had the highest concentration during the year (19 grains/m(3) per day; 27.5% of the total concentration of pollen). Tree pollen predominated from January through March; with Fraxinus spp, Morus spp, Celtis spp, Cupressus spp, and Pinus spp as the most important. Weed pollen predominated in May, June, and December and the most frequently identified, were Amaranthaceae/Chenopodiaceae, Ambrosia spp, and Parietaria spp. The highest concentration of grass pollen was reported during the months of May, June, September, October, and December with Gramineae/Poaceae predominating. Tree pollen was the most abundant during the year, with the ash tree having the highest concentration. Weed and grass pollen were perennial with peaks during the year.

  15. Pollen Forecast and Dispersion Modelling

    NASA Astrophysics Data System (ADS)

    Costantini, Monica; Di Giuseppe, Fabio; Medaglia, Carlo Maria; Travaglini, Alessandro; Tocci, Raffaella; Brighetti, M. Antonia; Petitta, Marcello

    2014-05-01

    The aim of this study is monitoring, mapping and forecast of pollen distribution for the city of Rome using in-situ measurements of 10 species of common allergenic pollens and measurements of PM10. The production of daily concentration maps, associated to a mobile phone app, are innovative compared to existing dedicated services to people who suffer from respiratory allergies. The dispersal pollen is one of the most well-known causes of allergic disease that is manifested by disorders of the respiratory functions. Allergies are the third leading cause of chronic disease and it is estimated that tens millions of people in Italy suffer from it. Recent works reveal that during the last few years there was a progressive increase of affected subjects, especially in urban areas. This situation may depend: on the ability to transport of pollutants, on the ability to react between pollutants and pollen and from a combination of other irritants, existing in densely populated and polluted urban areas. The methodology used to produce maps is based on in-situ measurements time series relative to 2012, obtained from networks of air quality and pollen stations in the metropolitan area of Rome. The monitoring station aerobiological of University of Rome "Tor Vergata" is located at the Department of Biology. The instrument used to pollen monitoring is a volumetric sampler type Hirst (Hirst 1952), Model 2000 VPPS Lanzoni; the data acquisition is carried out as reported in Standard UNI 11008:2004 - "Qualità dell'aria - Metodo di campionamento e conteggio dei granuli pollinici e delle spore fungine aerodisperse" - the protocol that describes the procedure for measuring of the concentration of pollen grains and fungal spores dispersed into the atmosphere, and reported in the "Manuale di gestione e qualità della R.I.M.A" (Travaglini et. al. 2009). All 10 allergenic pollen are monitored since 1996. At Tor Vergata university is also operating a meteorological station (SP2000, CAE

  16. Pollen Killer Gene S35 Function Requires Interaction with an Activator That Maps Close to S24, Another Pollen Killer Gene in Rice.

    PubMed

    Kubo, Takahiko; Yoshimura, Atsushi; Kurata, Nori

    2016-05-03

    Pollen killer genes disable noncarrier pollens, and are responsible for male sterility and segregation distortion in hybrid populations of distantly related plant species. The genetic networks and the molecular mechanisms underlying the pollen killer system remain largely unknown. Two pollen killer genes, S24 and S35, have been found in an intersubspecific cross of Oryza sativa ssp. indica and japonica The effect of S24 is counteracted by an unlinked locus EFS Additionally, S35 has been proposed to interact with S24 to induce pollen sterility. These genetic interactions are suggestive of a single S24-centric genetic pathway (EFS-S24-S35) for the pollen killer system. To examine this hypothetical genetic pathway, the S35 and the S24 regions were further characterized and genetically dissected in this study. Our results indicated that S35 causes pollen sterility independently of both the EFS and S24 genes, but is dependent on a novel gene close to the S24 locus, named incentive for killing pollen (INK). We confirmed the phenotypic effect of the INK gene separately from the S24 gene, and identified the INK locus within an interval of less than 0.6 Mb on rice chromosome 5. This study characterized the genetic effect of the two independent genetic pathways of INK-S35 and EFS-S24 in indica-japonica hybrid progeny. Our results provide clear evidence that hybrid male sterility in rice is caused by several pollen killer networks with multiple factors positively and negatively regulating pollen killer genes.

  17. A recombinant peptide model of the first nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator: comparison of wild-type and delta F508 mutant forms.

    PubMed Central

    Yike, I.; Ye, J.; Zhang, Y.; Manavalan, P.; Gerken, T. A.; Dearborn, D. G.

    1996-01-01

    A series of recombinant peptides, each including the sequence proposed to be the first nucleotide-binding fold of cystic fibrosis transmembrane conductance regulator (CFTR), has been produced in an attempt to find a model peptide that would autologously fold into a soluble structure with native-like properties. The peptide NBDIF, which contains the 267-amino acid sequence of CFTR from 384 to 650, meets these requirements. The peptide was produced with a high expression bacterial plasmid pRSET, purified from inclusion bodies following solubilization with 6 M guanidine-HCl and refolded from 8 M urea. Competitive displacement of trinitrophenol-ATP by nucleotides reveals binding of ATP and related nucleotides with KDs in the low micromolar range; the KD for ATP gamma S is 1.0 +/- 0.4 microM and for ADP 8.8 +/- 3.1 microM. The native-like character of the model peptide's structure is further supported by the findings that the KD for the ATP analog, 5'-adenylimidodiphosphate, is fourfold lower than the KD for the methylene analog, 5'-adenylmethylenediphosphonate, and that ATP binding slows the trypsin proteolysis of NBDIF. The CD spectra of NBDIF and the parallel peptide containing the most common cystic fibrosis mutation, deletion of Phe 508, are essentially indistinguishable, both spectra indicating 28% alpha-helix and 23% beta-sheet, with insignificant differences in the amounts of beta-turns and random structure. Extensive investigation using multiple conditions with highly purified preparations of the model peptides demonstrates that they do not support ATP hydrolysis. These large recombinant peptides offer practical models for the investigation of the first nucleotide-binding domain of CFTR. PMID:8771200

  18. Pollen mixing in pollen generalist solitary bees: a possible strategy to complement or mitigate unfavourable pollen properties?

    PubMed

    Eckhardt, Michael; Haider, Mare; Dorn, Silvia; Müller, Andreas

    2014-05-01

    Generalist herbivorous insects, which feed on plant tissue that is nutritionally heterogeneous or varies in its content of secondary metabolites, often benefit from dietary mixing through more balanced nutrient intake or reduced exposure to harmful secondary metabolites. Pollen is similarly heterogeneous as other plant tissue in its content of primary and secondary metabolites, suggesting that providing their offspring with mixed pollen diets might be a promising strategy for pollen generalist bees to complement nutrient imbalances or to mitigate harmful secondary metabolites of unfavourable pollen. In the present study, we compared larval performance of the pollen generalist solitary bee species Osmia cornuta (Megachilidae) on five experimental pollen diets that consisted of different proportions of unfavourable pollen diet of Ranunculus acris (Ranunculaceae) and favourable pollen diet of Sinapis arvensis (Brassicaceae). In addition, we microscopically analysed the pollen contained in the scopal brushes of field-collected females of O. cornuta and three closely related species to elucidate to what degree these pollen generalist bees mix pollen of different hosts in their brood cells. In striking contrast to a pure Ranunculus pollen diet, which had a lethal effect on most developing larvae of O. cornuta, larval survival, larval development time and adult body mass of both males and females remained nearly unaffected by the admixture of up to 50% of Ranunculus pollen diet to the larval food. Between 42% and 66% of all female scopal pollen loads analysed contained mixtures of pollen from two to six plant families, indicating that pollen mixing is a common behaviour in O. cornuta and the three related bee species. The present study provides the first evidence that the larvae of pollen generalist bees can benefit from the nutrient content of unfavourable pollen without being negatively affected by its unfavourable chemical properties if such pollen is mixed with

  19. Over-expression of miR158 causes pollen abortion in Brassica campestris ssp. chinensis.

    PubMed

    Ma, Zhiming; Jiang, Jianxia; Hu, Ziwei; Lyu, Tianqi; Yang, Yang; Jiang, Jingjing; Cao, Jiashu

    2017-02-01

    We identified and cloned the two precursors of miR158 and its target gene in Brassica campestris ssp. chinensis, which both had high relative expression in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility, which was caused by the degradation of pollen contents from the binucleate microspore stage. These results first suggest the role of miR158 in pollen development of Brassica campestris ssp. chinensis. MicroRNAs (miRNAs) play crucial roles in many important growth and development processes both in plants and animals by regulating the expression of their target genes via mRNA cleavage or translational repression. In this study, miR158, a Brassicaceae specific miRNA, was functionally characterized with regard to its role in pollen development of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). Two family members of miR158 in B. campestris, namely bra-miR158a1 and bra-miR158a2, and their target gene bra027656, which encodes a pentatricopeptide repeat (PPR) containing protein, were identified. Then, qRT-PCR analysis and GUS-reporter system revealed that both bra-miR158 and its target gene had relatively high expression levels in the inflorescences. Further study revealed that over-expression of miR158 caused reduced pollen varbility and pollen germination ratio, and the degradation of pollen contents from the binucleate microspore stage was also found in those deformed pollen grains, which led to pollen shrinking and collapse in later pollen development stage. These results first shed light on the importance of miR158 in pollen development of Brassica campestris ssp. chinensis.

  20. Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR

    PubMed Central

    Chambers, Carrie; Shuai, Bin

    2009-01-01

    Background MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. In higher plants, miRNAs are involved in developmental processes and stress responses. Sexual reproduction in flowering plants relies on pollen, the male gametophyte, to deliver sperm cells to fertilize the egg cell hidden in the embryo sac. Studies indicated that post-transcriptional processes are important for regulating gene expression during pollen function. However, we still have very limited knowledge on the involved gene regulatory mechanisms. Especially, the function of miRNAs in pollen remains unknown. Results Using miRCURY LNA array technology, we have profiled the expression of 70 known miRNAs (representing 121 miRBase IDs) in Arabidopsis mature pollen, and compared the expression of these miRNAs in pollen and young inflorescence. Thirty-seven probes on the array were identified using RNAs isolated from mature pollen, 26 of which showed significant differences in expression between mature pollen and inflorescence. Real-time PCR based on TaqMan miRNA assays confirmed the expression of 22 miRNAs in mature pollen, and identified 8 additional miRNAs that were expressed at low level in mature pollen. However, the expression of 11 miRNA that were identified on the array could not be confirmed by the Taqman miRNA assays. Analyses of transcriptome data for some miRNA target genes indicated that miRNAs are functional in pollen. Conclusion In summary, our results showed that some known miRNAs were expressed in Arabidopsis mature pollen, with most of them being low abundant. The results can be utilized in future research to study post-transcriptional gene regulation in pollen function. PMID:19591667

  1. Coconut (Cocos nucifera l.) pollen cryopreservation.

    PubMed

    Karun, A; Sjini, K K; Niral, V; Amarnth, C H; Remya, P; Rajesh, M K; Samsudeen, K; Jerard, B A; Engelmann, F

    2014-01-01

    Coconut genetic resources are threatened by pests and pathogens, natural hazards and human activities. Cryopreservation is the only method allowing the safe and cost-effective long-term conservation of recalcitrant seed species such as coconut. The objective of this work was to test the effect of cryopreservation and of cryostorage duration on coconut pollen germination and fertility. Pollen of two coconut varieties (West Coast Tall WWCTW and Chowghat Orange Dwarf CODC) was collected in March-May over three successive years, desiccated to 7.5 % moisture content (FW) and cryopreserved by direct immersion in liquid nitrogen. Germination and pollen tube length (PTL) of desiccated and cryopreserved pollen were not significantly different for both WCT and COD over the three harvest months of the three consecutive years of study. Pollen germination ranged from 24 to 32 % in desiccated pollen whereas it was between 26 and 29 % in cryopreserved COD pollen. In the case of WCT, germination ranged from 30 to 31 % in desiccated pollen, while it was between 28 and 32 % in cryopreserved pollen. PTL of cryopreserved pollen ranged between 224-390 nm and 226-396 mm for COD and WCT, respectively. Germination of COD pollen varied between 29.0 and 44.1 % after 4 years and 1.0/1.5 years cryostorage, respectively. Germination of WCT pollen did not change significantly between 0 and 6 years cryostorage, being comprised between 32 (24 h) and 40 % (1.5 years). Germination and vigour of cryopreserved pollen were generally higher compared to that of pollen dried in oven and non-cryopreserved. Normal seed set was observed in COD and WCT palms using pollen cryostored for 6 months and 4 years. Cryopreserved pollen of five Tall and five Dwarf accessions displayed 24-31 % and 25-49 % germination, respectively. These results show that it is now possible to establish pollen cryobanks to contribute to coconut germplasm long-term conservation.

  2. NaStEP: A Proteinase Inhibitor Essential to Self-Incompatibility and a Positive Regulator of HT-B Stability in Nicotiana alata Pollen Tubes1[W][OA

    PubMed Central

    Jiménez-Durán, Karina; McClure, Bruce; García-Campusano, Florencia; Rodríguez-Sotres, Rogelio; Cisneros, Jesús; Busot, Grethel; Cruz-García, Felipe

    2013-01-01

    In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown. PMID:23150644

  3. A new regulatory principle for in vivo biochemistry: pleiotropic low affinity regulation by the adenine nucleotides--illustrated for the glycolytic enzymes of Saccharomyces cerevisiae.

    PubMed

    Mensonides, Femke I C; Bakker, Barbara M; Cremazy, Frederic; Messiha, Hanan L; Mendes, Pedro; Boogerd, Fred C; Westerhoff, Hans V

    2013-09-02

    Enzymology tends to focus on highly specific effects of substrates, allosteric modifiers, and products occurring at low concentrations, because these are most informative about the enzyme's catalytic mechanism. We hypothesized that at relatively high in vivo concentrations, important molecular monitors of the state of living cells, such as ATP, affect multiple enzymes of the former and that these interactions have gone unnoticed in enzymology. We test this hypothesis in terms of the effect that ATP, ADP, and AMP might have on the major free-energy delivering pathway of the yeast Saccharomyces cerevisiae. Assaying cell-free extracts, we collected a comprehensive set of quantitative kinetic data concerning the enzymes of the glycolytic and the ethanol fermentation pathways. We determined systematically the extent to which the enzyme activities depend on the concentrations of the adenine nucleotides. We found that the effects of the adenine nucleotides on enzymes catalysing reactions in which they are not directly involved as substrate or product, are substantial. This includes effects on the Michaelis-Menten constants, adding new perspective on these, 100 years after their introduction.

  4. Comparative cytological and transcriptomic analysis of pollen development in autotetraploid and diploid rice.

    PubMed

    Wu, Jinwen; Shahid, Muhammad Qasim; Guo, Haibin; Yin, Wei; Chen, Zhixiong; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2014-12-01

    Autotetraploid rice has greater genetic variation and higher vigor than diploid rice, but low pollen fertility is one of the major reasons for low yield of autotetraploid rice. Very little is known about the molecular mechanisms of low pollen fertility of autotetraploid rice. In this study, cytological observations and microarray analysis were used to assess the genetic variation during pollen development in autotetraploid and diploid rice. Many abnormal chromosome behaviors, such as mutivalents, lagged chromosomes, asynchronous cell division, and so on, were found during meiosis in autotetraploid. Microsporogenesis and microgametogenesis in autotetraploid rice was similar to diploid rice, but many different kinds of abnormalities, including microspores degeneration, multi-aperture, and abnormal cell walls, were found in autotetraploid rice. Compared with diploid rice, a total of 1,251 genes were differentially expressed in autotetraploid rice in pollen transcriptome, among them 1,011 and 240 genes were up-regulated and down-regulated, respectively. 124 and 6 genes were co-up-regulated and co-down-regulated during three pollen development stages, respectively. These results suggest that polyploidy induced up-regulation for most of the genes during pollen development. Quantitative RT-PCR was done to validate 12 differentially expressed genes selected from functional categories based on the gene ontology analysis. These stably expressed genes not only related to the pollen development genes, but also involved in cell metabolism, cell physiology, binding, catalytic activity, molecular transducer activity, and transcription regulator activity. The present study suggests that differential expression of some key genes may lead to complex gene regulation and abnormal pollen development in autotetraploid rice.

  5. Advances in targeting cyclic nucleotide phosphodiesterases

    PubMed Central

    Maurice, Donald H.; Ke, Hengming; Ahmad, Faiyaz; Wang, Yousheng; Chung, Jay; Manganiello, Vincent C.

    2014-01-01

    Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants. PMID:24687066

  6. Plant cyclic nucleotide signalling: facts and fiction.

    PubMed

    Martinez-Atienza, Juliana; Van Ingelgem, Carl; Roef, Luc; Maathuis, Frans Jm

    2007-11-01

    The presence of the cyclic nucleotides 3',5'-cyclic adenyl monophosphate (cAMP) and 3',5'-cyclic guanyl monophosphate (cGMP) in plants is now generally accepted. In addition, cAMP and cGMP have been implicated in the regulation of important plant processes such as stomatal functioning, monovalent and divalent cation fluxes, chloroplast development, gibberellic acid signalling, pathogen response and gene transcription. However, very little is known regarding the components of cyclic nucleotide signalling in plants. In this addendum, the evidence for specific mechanisms of plant cyclic nucleotide signalling is evaluated and discussed.

  7. Vacuolar sorting receptors (VSRs) and secretory carrier membrane proteins (SCAMPs) are essential for pollen tube growth.

    PubMed

    Wang, Hao; Tse, Yu C; Law, Angus H Y; Sun, Samuel S M; Sun, Yong-Bin; Xu, Zeng-Fu; Hillmer, Stefan; Robinson, David G; Jiang, Liwen

    2010-03-01

    Vacuolar sorting receptors (VSRs) are type-I integral membrane proteins that mediate biosynthetic protein traffic in the secretory pathway to the vacuole, whereas secretory carrier membrane proteins (SCAMPs) are type-IV membrane proteins localizing to the plasma membrane and early endosome (EE) or trans-Golgi network (TGN) in the plant endocytic pathway. As pollen tube growth is an extremely polarized and highly dynamic process, with intense anterograde and retrograde membrane trafficking, we have studied the dynamics and functional roles of VSR and SCAMP in pollen tube growth using lily (Lilium longiflorum) pollen as a model. Using newly cloned lily VSR and SCAMP cDNA (termed LIVSR and LISCAMP, respectively), as well as specific antibodies against VSR and SCAMP1 as tools, we have demonstrated that in growing lily pollen tubes: (i) transiently expressed GFP-VSR/GFP-LIVSR is located throughout the pollen tubes, excepting the apical clear-zone region, whereas GFP-LISCAMP is mainly concentrated in the tip region; (ii) VSRs are localized to the multivesicular body (MVB) and vacuole, whereas SCAMPs are localized to apical endocytic vesicles, TGN and vacuole; and (iii) microinjection of VSR or SCAMP antibodies and LlVSR small interfering RNAs (siRNAs) significantly reduced the growth rate of the lily pollen tubes. Taken together, both VSR and SCAMP are required for pollen tube growth, probably working together in regulating protein trafficking in the secretory and endocytic pathways, which need to be coordinated in order to support pollen tube elongation.

  8. Secretory COPII Protein SEC31B Is Required for Pollen Wall Development1[OPEN

    PubMed Central

    Zhao, Bingchun; Shi, Haidan; Wang, Wanlei; Liu, Xiaoyu; Gao, Hui; Wang, Xiaoxiao; Zhang, Yinghui; Yang, Meidi; Li, Rui

    2016-01-01

    The pollen wall protects pollen grains from abiotic and biotic stresses. During pollen wall development, tapetal cells play a vital role by secreting proteins, signals, and pollen wall material to ensure microspore development. But the regulatory mechanism underlying the secretory pathway of the tapetum is largely unknown. Here, we characterize the essential role of the Arabidopsis (Arabidopsis thaliana) COPII protein SECRETORY31B (SEC31B) in pollen wall development and the secretory activity of tapetal cells. The sporophyte-controlled atsec31b mutant exhibits severe pollen and seed abortion. Transmission electron microscopy observation indicates that pollen exine formation in the atsec31b mutant is disrupted significantly. AtSEC31B is a functional COPII protein revealed by endoplasmic reticulum (ER) exit site localization, interaction with AtSEC13A, and retarded ER-Golgi protein trafficking in the atsec31b mutant. A genetic tapetum-specific rescue assay indicates that AtSEC31B functions primarily in the tapetum. Moreover, deletion of AtSEC31B interrupted the formation of the ER-derived tapetosome and altered the location of the ATP-BINDING CASSETTE TRANSPORTER9 protein in the tapetum. Therefore, this work demonstrates that AtSEC31B plays a vital role in pollen wall development by regulating the secretory pathway of the tapetal cells. PMID:27634427

  9. A cyclic nucleotide-gated channel is necessary for optimum fertility in high-calcium environments.

    PubMed

    Chaiwongsar, Suraphon; Strohm, Allison K; Roe, Joshua R; Godiwalla, Roxana Y; Chan, Catherine W M

    2009-01-01

    * Arabidopsis cngc2 plants are hypersensitive to external calcium and exhibit reduced plant size and fertility, especially when they are treated with elevated but physiologically relevant levels of calcium. This report focuses on the role of cyclic nucleotide-gated channel 2 (CNGC2) in plant fertility. * To determine the cause of the reduced fertility, we investigated the flower structure and growth potential of both male and female reproductive organs in cngc2 plants grown in high-calcium conditions. * cngc2 mutants had short stamens that may limit pollen deposition and pistils that were not conducive to pollen tube growth. * Our data indicate that sporophytic, but not gametophytic, defects are the main cause of the observed reduction in seed yield in cngc2 plants, and suggest that correct cyclic nucleotide and calcium signaling are important for cell elongation and pollen tube guidance.

  10. Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets†,‡

    PubMed Central

    Endrizzi, James A.; Kim, Hanseong; Anderson, Paul M.; Baldwin, Enoch P.

    2010-01-01

    Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-Å resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP- and UTP-binding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer–tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 Å between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. PMID:15157079

  11. Pollen Aquaporins: The Solute Factor

    PubMed Central

    Pérez Di Giorgio, Juliana A.; Soto, Gabriela C.; Muschietti, Jorge P.; Amodeo, Gabriela

    2016-01-01

    In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen’s success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange. PMID:27881985

  12. Does stronger pollen competition improve offspring fitness when pollen load does not vary?

    PubMed

    Pélabon, Christophe; Hennet, Lauriane; Bolstad, Geir H; Albertsen, Elena; Opedal, Øystein H; Ekrem, Runa K; Armbruster, W Scott

    2016-03-01

    Competition among pollen grains from a single donor is expected to increase the quality of the offspring produced because of the recessive deleterious alleles expressed during pollen-tube growth. However, evidence for such an effect is inconclusive; a large number of studies suffer from confounding variation in pollen competition with variation in pollen load. In this study, we tested the effect of pollen competition on offspring performance independently of pollen-load variation. We compared seed mass and early seedling performance in Dalechampia scandens (Euphorbiaceae) between crosses in which variation in pollen competition was achieved, without variation in pollen load, by manipulating the dispersion of pollen grains on the stigmas. Despite a large sample size (211 crosses on 20 maternal plants), we failed to find an effect of pollen competition on seed characteristics or early seedling performance. Paternal effects were always limited, and pollen competition never reduced the within-father (residual) variance. These results suggest that limited within-donor variation in genetic quality of pollen grains reduces the potential benefits of pollen competition in the study population. The lack of paternal effects on early sporophyte performance further suggests that benefits of pollen competition among pollen from multiple donors should be limited as well, and it raises questions about the significance of pollen competition as a mechanism of sexual selection. © 2016 Botanical Society of America.

  13. National Allergy Bureau Pollen and Mold Report

    MedlinePlus

    ... Search Search AAAAI National Allergy Bureau Pollen and Mold Report Date: April 12, 2017 Location: San Antonio ( ... Service can automatically email you daily pollen and mold reports. Click here sign up! Return to Map ...

  14. Bioassaying for ozone with pollen systems

    SciTech Connect

    Feder, W.A.

    1981-01-01

    Sensitivity to ozone of pollen germinating in vitro is closely correlated with ozone sensitivity of the pollen parent. Ozone-sensitive and tolerant pollen populations have been identified in tobacco, petunia, and tomato cultivars. The rate of tube elongation can be reversibly slowed or stopped by exposure to low concentrations of ozone. The performance of selected pollen populations can then be used to bioassay ozone in ambient air by introducing the air sample into a growth chamber where ozone-sensitive pollen in growing. Year-round pollen producion can be achieved in the greenhouse. Harvested pollen can be tested, packaged, and transported to user facilities without loss of vigor. Pollen populations are inexpensive to produce, respond reliably, and are simple to use as a bioassay for air quality.

  15. Pollen aquaporins: What are they there for?

    PubMed

    Pérez Di Giorgio, Juliana Andrea; Barberini, María Laura; Amodeo, Gabriela; Muschietti, Jorge Prometeo

    2016-09-01

    In order to provide more insight into the function of aquaporins during pollination, we characterized NIP4;1 and NIP4;2, 2 pollen-specific aquaporins of Arabidopsis thaliana. NIP4;1 and NIP4;2 displayed high amino acid identity. RT-PCR and GUS promoter analysis showed that they have different expression patterns. NIP4;1 is expressed at low levels in mature pollen, while NIP4;2 is highly expressed only during pollen tube growth. Single T-DNA nip4;1 and nip4;2 mutants and double amiRNA nip4;1 nip4;2 knockdowns showed reduced male fertility due to deficient pollen germination and pollen tube length. Functional assays in oocytes showed that NIP4;1 and NIP4;2 transport water and nonionic solutes. Here, the participation of the different pollen aquaporins in pollen hydration and pollen tube growth is discussed.

  16. Shifting Weather Can Unleash Pollen 'Superburst'

    MedlinePlus

    ... 166183.html Shifting Weather Can Unleash Pollen 'Superburst' Trees, grasses and weeds may bloom all at once, ... news release. Usually, pollens come in waves. First, trees, then weeds, then grasses. But this year, they ...

  17. [The epidemiology of pollen allergy].

    PubMed

    Charpin, D; Caillaud, D

    2014-04-01

    The prevalence of seasonal allergic rhinitis can be established through surveys performed in a sample of the general population. These surveys are based on a questionnaire, which could lead to an overestimate of prevalence rates, and on measurements of specific IgE, which need to be interpreted in the light of the responses to the questionnaire. Such surveys are few in France and need to be updated. Risk factors for seasonal allergic rhinitis are genetic, epigenetic and environmental. Relationships between exposure to pollen and health can be documented through ecological and panel surveys. Panel surveys may give information on threshold levels and dose-response relationships. In addition to pollen exposure, global warming and air pollutants act as cofactors. Monitoring of both pollen exposure and its health effects should be encouraged and strengthened.

  18. Pollen Allergens for Molecular Diagnosis.

    PubMed

    Pablos, Isabel; Wildner, Sabrina; Asam, Claudia; Wallner, Michael; Gadermaier, Gabriele

    2016-04-01

    Pollen allergens are one of the main causes of type I allergies affecting up to 30% of the population in industrialized countries. Climatic changes affect the duration and intensity of pollen seasons and may together with pollution contribute to increased incidences of respiratory allergy and asthma. Allergenic grasses, trees, and weeds often present similar habitats and flowering periods compromising clinical anamnesis. Molecule-based approaches enable distinction between genuine sensitization and clinically mostly irrelevant IgE cross-reactivity due to, e. g., panallergens or carbohydrate determinants. In addition, sensitivity as well as specificity can be improved and lead to identification of the primary sensitizing source which is particularly beneficial regarding polysensitized patients. This review gives an overview on relevant pollen allergens and their usefulness in daily practice. Appropriate allergy diagnosis is directly influencing decisions for therapeutic interventions, and thus, reliable biomarkers are pivotal when considering allergen immunotherapy in the context of precision medicine.

  19. Pine pollens frozen five years produce seed

    Treesearch

    R.Z. Callaham; R.J. Steinhoff

    1966-01-01

    Deep-freezing of pine pollen offers a means of prolonging its storage life. Early work showed that pollen could be frozen without losing its viability. A study was started in 1958 at the Institute of Forest Genetics at Placerville to determine how long frozen pollen of several pines would remain viable. This paper reports in vitro germination and in vivo seed...

  20. Overexpression of the Tomato Pollen Receptor Kinase LePRK1 Rewires Pollen Tube Growth to a Blebbing Mode[W][OPEN

    PubMed Central

    Gui, Cai-Ping; Dong, Xin; Liu, Hai-Kuan; Huang, Wei-Jie; Zhang, Dong; Wang, Shu-Jie; Barberini, María Laura; Gao, Xiao-Yan; Muschietti, Jorge; McCormick, Sheila

    2014-01-01

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP), a Rop guanine nucleotide exchange factor. Here, we show that pollen tubes overexpressing LePRK1 or a truncated LePRK1 lacking its extracellular domain (LePRK1ΔECD) have enlarged tips but also extend their leading edges by producing “blebs.” Coexpression of LePRK1 and tomato PLIM2a, an actin bundling protein that interacts with KPP in a Ca2+-responsive manner, suppressed these LePRK1 overexpression phenotypes, whereas pollen tubes coexpressing KPP, LePRK1, and PLIM2a resumed the blebbing growth mode. We conclude that overexpression of LePRK1 or LePRK1ΔECD rewires pollen tube growth to a blebbing mode, through KPP- and PLIM2a-mediated bundling of actin filaments from tip plasma membranes. Arabidopsis thaliana pollen tubes expressing LePRK1ΔECD also grew by blebbing. Our results exposed a hidden capability of the pollen tube cell: upon overexpression of a single membrane-localized molecule, LePRK1 or LePRK1ΔECD, it can switch to an alternative mechanism for extension of the leading edge that is analogous to the blebbing growth mode reported for Dictyostelium and for Drosophila melanogaster stem cells. PMID:25194029

  1. Addition of rye chromosome 4R to wheat increases anther length and pollen grain number.

    PubMed

    Nguyen, Vy; Fleury, Delphine; Timmins, Andy; Laga, Hamid; Hayden, Matthew; Mather, Diane; Okada, Takashi

    2015-05-01

    The research identified rye chromosome 4R arms associated with good pollinator traits, and demonstrated possible use of rye genetic resources to develop elite pollinators for hybrid wheat breeding. Bread wheat (Triticum aestivum) is a predominantly self-pollinating plant which has relatively small-sized anthers and produces a low number of pollen grains. These features limit the suitability of most wheat lines as pollinators for hybrid seed production. One strategy for improving the pollination ability of wheat is to introgress cross-pollination traits from related species. One such species is rye (Secale cereale L.), which has suitable traits such as high anther extrusion, long anthers containing large amounts of pollen and long pollen viability. Therefore, introducing these traits into wheat is of great interest in hybrid wheat breeding. Here, we investigated wheat-rye chromosome addition lines for the effects of rye chromosomes on anther and pollen development in wheat. Using a single nucleotide polymorphism genotyping array, we detected 984 polymorphic markers that showed expected syntenic relationships between wheat and rye. Our results revealed that the addition of rye chromosomes 1R or 2R reduced pollen fertility, while addition of rye chromosome 4R increased anther size by 16% and pollen grain number by 33%. The effect on anther length was associated with increases in both cell size and the number of endothecium cells and was attributed to the long arm of chromosome 4R. In contrast, the effect on pollen grain number was attributed to the short arm of chromosome 4R. These results indicate that rye chromosome 4R contains at least two genetic factors associated with increased anther size and pollen grain number that can favourably affect pollination traits in wheat.

  2. Rad and Rem are non-canonical G-proteins with respect to the regulatory role of guanine nucleotide binding in Ca(V)1.2 channel regulation.

    PubMed

    Chang, Donald D; Colecraft, Henry M

    2015-12-01

    Rad and Rem are Ras-like G-proteins linked to diverse cardiovascular functions and pathophysiology. Understanding how Rad and Rem are regulated is important for deepened insights into their pathophysiological roles. As in other Ras-like G-proteins, Rad and Rem contain a conserved guanine-nucleotide binding domain (G-domain). Canonically, G-domains are key control modules, functioning as nucleotide-regulated switches of G-protein activity. Whether Rad and Rem G-domains conform to this canonical paradigm is ambiguous. Here, we used multiple functional measurements in HEK293 cells and cardiomyocytes (Ca(V)1.2 currents, Ca(2+) transients, Ca(V)β binding) as biosensors to probe the role of the G-domain in regulation of Rad and Rem function. We utilized Rad(S105N) and Rem(T94N), which are the cognate mutants to Ras(S17N), a dominant-negative variant of Ras that displays decreased nucleotide binding affinity. In HEK293 cells, over-expression of either Rad(S105N) or Rem(T94N) strongly inhibited reconstituted Ca(V)1.2 currents to the same extent as their wild-type (wt) counterparts, contrasting with reports that Rad(S105N) is functionally inert in HEK293 cells. Adenovirus-mediated expression of either wt Rad or Rad(S105N) in cardiomyocytes dramatically blocked L-type calcium current (I(Ca,L)) and inhibited Ca(2+)-induced Ca(2+) release, contradicting reports that Rad(S105N) acts as a dominant negative in heart. By contrast, Rem(T94N) was significantly less effective than wt Rem at inhibiting I(Ca,L) and Ca(2+) transients in cardiomyocytes. FRET analyses in cardiomyocytes revealed that both Rad(S105N) and Rem(T94N) had moderately reduced binding affinity for Ca(V)βs relative to their wt counterparts. The results indicate Rad and Rem are non-canonical G-proteins with respect to the regulatory role of their G-domain in Ca(V)1.2 regulation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  3. A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation1[OPEN

    PubMed Central

    Yu, Jing; Meng, Zhaolu; Behera, Smrutisanjita; Kudla, Jörg; Luo, Zhijing; Wang, Jie; Zhang, Siyi

    2016-01-01

    In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca2+ is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca2+ binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca2+ and regulate Ca2+ homeostasis in vitro, and osdex1 exhibited disturbed Ca2+ homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca2+ in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca2+ homeostasis during pollen development. PMID:27663411

  4. A Rice Ca2+ Binding Protein Is Required for Tapetum Function and Pollen Formation.

    PubMed

    Yu, Jing; Meng, Zhaolu; Liang, Wanqi; Behera, Smrutisanjita; Kudla, Jörg; Tucker, Matthew R; Luo, Zhijing; Chen, Mingjiao; Xu, Dawei; Zhao, Guochao; Wang, Jie; Zhang, Siyi; Kim, Yu-Jin; Zhang, Dabing

    2016-11-01

    In flowering plants, successful male reproduction requires the sophisticated interaction between somatic anther wall layers and reproductive cells. Timely degradation of the innermost tissue of the anther wall layer, the tapetal layer, is critical for pollen development. Ca(2+) is a well-known stimulus for plant development, but whether it plays a role in affecting male reproduction remains elusive. Here we report a role of Defective in Exine Formation 1 (OsDEX1) in rice (Oryza sativa), a Ca(2+) binding protein, in regulating rice tapetal cell degradation and pollen formation. In osdex1 anthers, tapetal cell degeneration is delayed and degradation of the callose wall surrounding the microspores is compromised, leading to aborted pollen formation and complete male sterility. OsDEX1 is expressed in tapetal cells and microspores during early anther development. Recombinant OsDEX1 is able to bind Ca(2+) and regulate Ca(2+) homeostasis in vitro, and osdex1 exhibited disturbed Ca(2+) homeostasis in tapetal cells. Phylogenetic analysis suggested that OsDEX1 may have a conserved function in binding Ca(2+) in flowering plants, and genetic complementation of pollen wall defects of an Arabidopsis (Arabidopsis thaliana) dex1 mutant confirmed its evolutionary conservation in pollen development. Collectively, these findings suggest that OsDEX1 plays a fundamental role in the development of tapetal cells and pollen formation, possibly via modulating the Ca(2+) homeostasis during pollen development.

  5. The proteome and phosphoproteome of maize pollen uncovers fertility candidate proteins.

    PubMed

    Chao, Qing; Gao, Zhi-Fang; Wang, Yue-Feng; Li, Zhe; Huang, Xia-He; Wang, Ying-Chun; Mei, Ying-Chang; Zhao, Biligen-Gaowa; Li, Liang; Jiang, Yu-Bo; Wang, Bai-Chen

    2016-06-01

    Maize is unique since it is both monoecious and diclinous (separate male and female flowers on the same plant). We investigated the proteome and phosphoproteome of maize pollen containing modified proteins and here we provide a comprehensive pollen proteome and phosphoproteome which contain 100,990 peptides from 6750 proteins and 5292 phosphorylated sites corresponding to 2257 maize phosphoproteins, respectively. Interestingly, among the total 27 overrepresented phosphosite motifs we identified here, 11 were novel motifs, which suggested different modification mechanisms in plants compared to those of animals. Enrichment analysis of pollen phosphoproteins showed that pathways including DNA synthesis/chromatin structure, regulation of RNA transcription, protein modification, cell organization, signal transduction, cell cycle, vesicle transport, transport of ions and metabolisms, which were involved in pollen development, the following germination and pollen tube growth, were regulated by phosphorylation. In this study, we also found 430 kinases and 105 phosphatases in the maize pollen phosphoproteome, among which calcium dependent protein kinases (CDPKs), leucine rich repeat kinase, SNF1 related protein kinases and MAPK family proteins were heavily enriched and further analyzed. From our research, we also uncovered hundreds of male sterility-associated proteins and phosphoproteins that might influence maize productivity and serve as targets for hybrid maize seed production. At last, a putative complex signaling pathway involving CDPKs, MAPKs, ubiquitin ligases and multiple fertility proteins was constructed. Overall, our data provides new insight for further investigation of protein phosphorylation status in mature maize pollen and construction of maize male sterile mutants in the future.

  6. MALE STERILITY1 Is Required for Tapetal Development and Pollen Wall Biosynthesis[W][OA

    PubMed Central

    Yang, Caiyun; Vizcay-Barrena, Gema; Conner, Katie; Wilson, Zoe A.

    2007-01-01

    The Arabidopsis thaliana MALE STERILITY1 (MS1) gene is critical for viable pollen formation and has homology to the PHD-finger class of transcription factors; however, its role in pollen development has not been fully defined. We show that MS1 transcription appears to be autoregulated by the wild-type MS1 transcript or protein. Using a functional green fluorescent protein (GFP) fusion to analyze the temporal and spatial expression of MS1, we demonstrate that the MS1:GFP protein is nuclear localized within the tapetum and is expressed in a developmentally regulated manner between late tetraspore and microspore release, then rapidly breaks down, probably by ubiquitin-dependent proteolysis. Absence of MS1 expression results in changes in tapetal secretion and exine structure. Microarray analysis has shown that 260 (228 downregulated and 32 upreglated) genes have altered expression in young ms1 buds. These genes are primarily associated with pollen wall and coat formation; however, a number of transcription factors and Cys proteases have also been identified as the putative primary regulatory targets of MS1. Ectopic expression of MS1 alters transcriptional regulation of vegetative gene expression, resulting in stunted plants with increased levels of branching, partially fertile flowers and an apparent increase in wall material on mature pollen. MS1 therefore plays a critical role in the induction of pollen wall and pollen coat materials in the tapetum and, ultimately, the production of viable pollen. PMID:18032629

  7. Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize

    PubMed Central

    Dong, Xue; Wang, Dongxue; Liu, Peng; Li, Chengxia; Zhao, Qian; Zhu, Dengyun; Yu, Jingjuan

    2013-01-01

    Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription–PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein–protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize. PMID:23676884

  8. BURSTING POLLEN is required to organize the pollen germination plaque and pollen tube tip in Arabidopsis thaliana.

    PubMed

    Hoedemaekers, Karin; Derksen, Jan; Hoogstrate, Suzanne W; Wolters-Arts, Mieke; Oh, Sung-Aeong; Twell, David; Mariani, Celestina; Rieu, Ivo

    2015-04-01

    Pollen germination may occur via the so-called germination pores or directly through the pollen wall at the site of contact with the stigma. In this study, we addressed what processes take place during pollen hydration (i.e. before tube emergence), in a species with extra-poral pollen germination, Arabidopsis thaliana. A T-DNA mutant population was screened by segregation distortion analysis. Histological and electron microscopy techniques were applied to examine the wild-type and mutant phenotypes. Within 1 h of the start of pollen hydration, an intine-like structure consisting of cellulose, callose and at least partly de-esterified pectin was formed at the pollen wall. Subsequently, this 'germination plaque' gradually extended and opened up to provide passage for the cytoplasm into the emerging pollen tube. BURSTING POLLEN (BUP) was identified as a gene essential for the correct organization of this plaque and the tip of the pollen tube. BUP encodes a novel Golgi-located glycosyltransferase related to the glycosyltransferase 4 (GT4) subfamily which is conserved throughout the plant kingdom. Extra-poral pollen germination involves the development of a germination plaque and BUP defines the correct plastic-elastic properties of this plaque and the pollen tube tip by affecting pectin synthesis or delivery.

  9. Characterization of a caleosin expressed during olive (Olea europaea L.) pollen ontogeny

    PubMed Central

    2011-01-01

    Background The olive tree is an oil-storing species, with pollen being the second most active site in storage lipid biosynthesis. Caleosins are proteins involved in storage lipid mobilization during seed germination. Despite the existence of different lipidic structures in the anther, there are no data regarding the presence of caleosins in this organ to date. The purpose of the present work was to characterize a caleosin expressed in the olive anther over different key stages of pollen ontogeny, as a first approach to unravel its biological function in reproduction. Results A 30 kDa caleosin was identified in the anther tissues by Western blot analysis. Using fluorescence and transmission electron microscopic immunolocalization methods, the protein was first localized in the tapetal cells at the free microspore stage. Caleosins were released to the anther locule and further deposited onto the sculptures of the pollen exine. As anthers developed, tapetal cells showed the presence of structures constituted by caleosin-containing lipid droplets closely packed and enclosed by ER-derived cisternae and vesicles. After tapetal cells lost their integrity, the caleosin-containing remnants of the tapetum filled the cavities of the mature pollen exine, forming the pollen coat. In developing microspores, this caleosin was initially detected on the exine sculptures. During pollen maturation, caleosin levels progressively increased in the vegetative cell, concurrently with the number of oil bodies. The olive pollen caleosin was able to bind calcium in vitro. Moreover, PEGylation experiments supported the structural conformation model suggested for caleosins from seed oil bodies. Conclusions In the olive anther, a caleosin is expressed in both the tapetal and germ line cells, with its synthesis independently regulated. The pollen oil body-associated caleosin is synthesized by the vegetative cell, whereas the protein located on the pollen exine and its coating has a sporophytic

  10. Evolving nucleotide binding surfaces

    NASA Technical Reports Server (NTRS)

    Kieber-Emmons, T.; Rein, R.

    1981-01-01

    An analysis is presented of the stability and nature of binding of a nucleotide to several known dehydrogenases. The employed approach includes calculation of hydrophobic stabilization of the binding motif and its intermolecular interaction with the ligand. The evolutionary changes of the binding motif are studied by calculating the Euclidean deviation of the respective dehydrogenases. Attention is given to the possible structural elements involved in the origin of nucleotide recognition by non-coded primordial polypeptides.

  11. Regulation of follitropin-sensitive adenylate cyclase by stimulatory and inhibitory forms of the guanine nucleotide regulatory protein in immature rat Sertoli cells

    SciTech Connect

    Johnson, G.P.

    1987-01-01

    Studies have been designed to examine the role of guanine nucleotides in mediating FSH-sensitive adenylate cyclase activity in Sertoli cell plasma membranes. Analysis of ({sup 3}H)GDP binding to plasma membranes suggested a single high affinity site with a K{sub d} = 0.24 uM. Competition studies indicated that GTP{sub {gamma}}S was 7-fold more potent than GDP{sub {beta}}S. Bound GDP could be released by FSH in the presence of GTP{sub {gamma}}S, but not by FSH alone. Adenylate cyclase activity was enhanced 5-fold by FSH in the presence of GTP. Addition of GDP{sub {beta}}S to the activated enzyme (FSH plus GTP) resulted in a time-dependent decay to basal activity within 20 sec. GDP{sub {beta}}S competitively inhibited GTP{sub {gamma}}S-stimulated adenylate cyclase activity with a K{sub i} = 0.18 uM. Adenylate cyclase activity was also demonstrated to be sensitive to the nucleotide bound state. In the presence of FSH, only the GTP{sub {gamma}}S-bound form persisted even if GDP{sub {beta}}S previously occupied all available binding sites. Two membrane proteins, M{sub r} = 43,000 and 48,000, were ADP{centered dot}ribosylated using cholera toxin and labeling was enhanced 2 to 4-fold by GTP{sub {gamma}}S but not by GDP{sub {beta}}S. The M{sub r} = 43,000 and 48,000 proteins represented variant forms of G{sub S}. A single protein of M{sub r} = 40,000 (G{sub i}) was ADP-ribosylated by pertussis toxin in vitro. GTP inhibited forskolin-stimulated adenylate cyclase activity with an IC{sub 50} = 0.1 uM. The adenosine analog, N{sup 6}{centered dot}phenylisopropyl adenosine enhanced GTP inhibition of forskolin-stimulated adenylate cyclase activity by an additional 15%. GTP-dependent inhibition of forskolin-sensitive adenylate cyclase activity was abolished in membranes prepared from Sertoli cells treated in culture with pertussis toxin.

  12. A group-1 grass pollen allergen influences the outcome of pollen competition in maize.

    PubMed

    Valdivia, Elene R; Wu, Yajun; Li, Lian-Chao; Cosgrove, Daniel J; Stephenson, Andrew G

    2007-01-17

    Worldwide, 400 million people suffer from hay fever and seasonal asthma. The major causative agents of these allergies are pollen specific proteins called the group-1 grass pollen allergens. Although details of their antigenicity have been studied for 40 years with an eye towards immunotherapy, their function in the plant has drawn scant attention. Zea m 1 constitutes a class of abundant grass pollen allergens coded for by several genes that loosen the walls of grass cells, including the maize stigma and style. We have examined the impact of a transposon insertion into one of these genes (EXPB1, the most abundant isoform of Zea m 1) on the production of Zea m 1 protein, pollen viability, and pollen tube growth, both in vitro and in vivo. We also examined the effect of the insertional mutation on the competitive ability of the pollen by experimentally varying the sizes of the pollen load deposited onto stigmas using pollen from heterozygous plants and then screening the progeny for the presence of the transposon using PCR. We found that the insertional mutation reduced the levels of Zea m 1 in maize pollen, but had no effect on pollen viability, in vitro pollen tube growth or the proportion of progeny sired when small pollen loads are deposited onto stigmas. However, when large pollen loads are deposited onto the stigmas, the transposon mutation is vastly underrepresented in the progeny, indicating that this major pollen allergen has a large effect on pollen tube growth rates in vivo, and plays an important role in determining the outcome of the pollen-pollen competition for access to the ovules. We propose that the extraordinary abundance (4% of the extractable protein in maize pollen) of this major pollen allergen is the result of selection for a trait that functions primarily in providing differential access to ovules.

  13. Proteomics of pollen development and germination.

    PubMed

    Dai, Shaojun; Wang, Tai; Yan, Xiufeng; Chen, Sixue

    2007-12-01

    In higher plants, pollen grains represent the vestiges of a highly reduced male gametophyte generation. After germination, the pollen tube delivers the sperm cells by tip-growing to the embryo sac for fertilization. Besides the intrinsic importance for sexual reproduction, pollen development and germination serve as an attractive system to address important questions related to cell division, cell differentiation, polar growth, cell-cell interaction, and cell fate. Recently, pollen functional specification has been well-studied using multidisciplinary approaches. Here, we review recent advances in proteomics of pollen development and germination.

  14. The nucleotide sequence of the nitrogen-regulation gene ntrA of Klebsiella pneumoniae and comparison with conserved features in bacterial RNA polymerase sigma factors.

    PubMed Central

    Merrick, M J; Gibbins, J R

    1985-01-01

    The nucleotide sequence of the Klebsiella pneumoniae ntrA gene has been determined. NtrA encodes a 53,926 Dalton acidic polypeptide; a calculated molecular weight which is significantly lower than that determined by SDS polyacrylamide gel analysis. NtrA is followed by another open-reading frame (orf) of at least 75 amino acids. In the spacer region between ntrA and orf there are no apparent transcription termination or promoter sequences and therefore orf may be co-transcribed with ntrA. Previous authors have proposed that NtrA could act as an RNA polymerase sigma factor but the NtrA amino acid sequence does not show a high level of homology to any known sigma factor. However analysis of sequences of five sigma factors from E. coli and B. subtilis has identified two conserved sequences at the C-terminal end of all these polypeptides. These sequences resemble those found in known site-specific DNA-binding domains and may be involved in recognition of conserved -35 and -10 promoter sequences. A similar pair of sequences is present at the C-terminus of NtrA and could play a role in recognition of ntr-activatable promoters. Images PMID:2999700

  15. Identification of a single nucleotide promoter polymorphism regulating the transcription of ubiquitin specific protease 18 gene related to the resistance to porcine reproductive and respiratory syndrome virus infection.

    PubMed

    Li, Yanping; Sun, Yi; Xing, Feng; Kang, Li; Wang, Pengfei; Wang, Liyuan; Liu, Hao; Li, Yi; Jiang, Yunliang

    2014-12-15

    Porcine reproductive and respiratory syndrome (PRRS), characterized by reproductive failure in sows and respiratory disease and mortality in piglets, is a major infectious disease that causes great economic loss throughout the world. Previous studies revealed that the overexpression of porcine ubiquitin specific protease 18 (USP18) gene inhibits PRRSV replication in vitro. The objective of this study is to compare the promoter activity of USP18 in Chinese indigenous Dapulian (DPL) pigs and Duroc×Landrace×Yorkshire (DLY) commercial pigs and screen single nucleotide polymorphism (SNP) affecting porcine USP18 transcription. We found that the promoter activity was significantly higher in DPL pigs than DLY commercial pigs (p<0.05), deletion of the promoter from -1790 to -1314bp decreased the transcriptional activity by roughly 60% (p<0.05) and a SNP G-1533A in this region increased the mRNA expression both prior to and post PRRSV infection in MARC-145 cells. Population genetics analysis showed that allele A was only detected in Chinese pig breeds which are generally resistant to PRRSV. These results suggest that the SNP G-1533A polymorphism in the promoter region of porcine USP18 gene is a potential DNA marker for the resistance to PRRSV. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Measuring Meiotic Crossovers via Multi-Locus Genotyping of Single Pollen Grains in Barley.

    PubMed

    Dreissig, Steven; Fuchs, Jörg; Cápal, Petr; Kettles, Nicola; Byrne, Ed; Houben, Andreas

    2015-01-01

    The detection of meiotic crossovers in crop plants currently relies on scoring DNA markers in a segregating population or cytological visualization. We investigated the feasibility of using flow-sorted haploid nuclei, Phi29 DNA polymerase-based whole-genome-amplification (WGA) and multi-locus KASP-genotyping to measure meiotic crossovers in individual barley pollen grains. To demonstrate the proof of concept, we used 24 gene-based physically mapped single nucleotide polymorphisms to genotype the WGA products of 50 single pollen nuclei. The number of crossovers per chromosome, recombination frequencies along chromosome 3H and segregation distortion were analysed and compared to a doubled haploid (DH) population of the same genotype. The number of crossovers and chromosome wide recombination frequencies show that this approach is able to produce results that resemble those obtained from other methods in a biologically meaningful way. Only the segregation distortion was found to be lower in the pollen population than in DH plants.

  17. A Simple, Inexpensive Pollen Trap

    Treesearch

    P. E. Hoekstra

    1965-01-01

    Pollen plays a role of vital importance in the sexual reproduction of all plants but it is especially important in forestry. With few exceptions, sexual reproduction is the only link between succeeding generations in the forest. To be sure, vegetative reproduction is important for special purposes, but it will probably not be used on a mass scale in timber...

  18. Bioassaying for ozone with pollen systems.

    PubMed Central

    Feder, W A

    1981-01-01

    Sensitivity to ozone of pollen germinating in vitro is closely correlated with ozone sensitivity of the pollen parent. Ozone-sensitive and tolerant pollen populations have been identified in tobacco, petunia, and tomato cultivars. The rate of tube elongation can be reversibly slowed or stopped by exposure to low concentrations of ozone. Tube growth rates in the presence of a range of ozone dosages, of pollen populations exhibiting differing ozone sensitivity can be measured and different growth rates can be correlated with ozone dosages. The performance of selected pollen populations can then be used to bioassay ozone in ambient air by introducing the air sample into a growth chamber where ozone-sensitive pollen in growing. Petunia and tobacco pollen are especially useful because they store well at ordinary freezer temperatures and do not require special preparation prior to storage. Modified Brewbacker's growth medium is suitable for growth of both these pollen types. Four useful cultivars are Bel W-3, ozone-sensitive and Bel B, ozone-tolerant tobacco, and White Bountiful, ozone-sensitive and Blue Lagoon, ozone-tolerant petunia. Observations can be made directly by using a TV scanner, or by time lapse or interval photography. Year-round pollen production can be achieved in the greenhouse. Harvested pollen can be tested, packaged, and transported to user facilities without loss of vigor. Pollen populations are inexpensive to produce, respond reliably, and are simple to use as a bioassay for air quality. Images FIGURE 2. FIGURE 3. FIGURE 4. PMID:7460876

  19. Brassinosteroids promote Arabidopsis pollen germination and growth.

    PubMed

    Vogler, Frank; Schmalzl, Christina; Englhart, Maria; Bircheneder, Martin; Sprunck, Stefanie

    2014-09-01

    Pollen tubes are among the fastest tip-growing plant cells and represent an excellent experimental system for studying the dynamics and spatiotemporal control of polarized cell growth. However, investigating pollen tube tip growth in the model plant Arabidopsis remains difficult because in vitro pollen germination and pollen tube growth rates are highly variable and largely different from those observed in pistils, most likely due to growth-promoting properties of the female reproductive tract. We found that in vitro grown Arabidopsis pollen respond to brassinosteroid (BR) in a dose-dependent manner. Pollen germination and pollen tube growth increased nine- and fivefold, respectively, when media were supplemented with 10 µM epibrassinolide (epiBL), resulting in growth kinetics more similar to growth in vivo. Expression analyses show that the promoter of one of the key enzymes in BR biosynthesis, CYP90A1/CPD, is highly active in the cells of the reproductive tract that form the pathway for pollen tubes from the stigma to the ovules. Pollen tubes grew significantly shorter through the reproductive tract of a cyp90a1 mutant compared to the wild type, or to a BR perception mutant. Our results show that epiBL promotes pollen germination and tube growth in vitro and suggest that the cells of the reproductive tract provide BR compounds to stimulate pollen tube growth.

  20. Cytochemical localization of some hydrolases in the pollen and pollen tubes of Amaryllis vittata Ait.

    PubMed

    Sharma, D

    1982-01-01

    Some hydrolases are localized cytochemically in the pollen and pollen tubes of Amaryllis vittata Ait. The function of different enzymes is discussed in relation to pollen tubes morphogenesis. Activity of most of the enzymes was confined to colpus region, pollen wall and general cytoplasm of pollen and pollen tube. The activity of hydrolytic enzymes like acid monophosphoesterase and lipase and was nil in the exine of both germinated and ungerminated pollen, whereas intense reaction for esterase was observed in exine. Enzyme activity increased after germination which suggest the hydrolysis of stored metabolites and synthesis of proteins and other metabolites for the active growth of pollen tube. Intense reaction for enzymes like alkaline phosphomonoesterase, ATP-ase, 5-nucleotidase etc. at the tip region of pollen tube suggest their role in physiological processes associated with exchange of materials through intercellular transport during tube wall polysaccharide biogenesis.

  1. Pollen resistance to water in 80 angiosperm species: flower structures protect rain-susceptible pollen.

    PubMed

    Mao, Yun-Yun; Huang, Shuang-Quan

    2009-08-01

    Flowers exhibit adaptive responses to biotic and abiotic factors. It remains unclear whether pollen susceptibility to rain damage plays a role in the evolution of floral form. We investigated flower performance in rain and compared pollen longevity in dry conditions, pure water and solutions with different sucrose concentrations in 80 flowering species from 46 families with diverse floral shapes and pollination modes. A pollen viability test showed that pollen longevity in all studied species was greatly reduced by wetting. We found that pollen of species with complete protection by flower structures was susceptible to water damage and a high proportion of resistant pollen occurred in unprotected species. Flowers whose structures expose pollen to rain may also reduce rain damage through temporal patterns of pollen presentation. This prediction was supported by our direct measurement of pollen presentation duration on rainy days. Our observations showed that variation in pollen performance in water was associated with differences in floral forms. Water-resistant pollen and extended pollen presentation duration were favored by selection via rain contact in species in which pollen was not protected from rain. These findings support the functional hypothesis that flower structures protect susceptible pollen from rain, demonstrating that rain acts as a force shaping floral form.

  2. Pollen selection under acid rain stress

    SciTech Connect

    Zhang, Y.

    1994-01-01

    To investigate whether acid rain stress induces pollen selection in nature, three different approaches were used, based on the assumption that the response of pollen grains to acid rain is controlled by an acid sensitive gene product. Germination of pollen from homozygous and heterozygous individuals under acid rain stress was examined to detect any differences in rate of germination between populations of homogeneous and heterogeneous pollen grains. In vitro and in vivo bulked segregant analysis using RAPDs was used to search for differences in DNA constitution between the survivors of acid rain stressed and non-acid rain stressed pollen populations in vitro and between the progenies of acid rain stressed and non-acid rain stressed populations during pollination, respectively. No evidence for the pollen selection under acid rain stress was obtained in any of the test systems. Inhibition of protein synthesis using cycloheximide led to significant reduction of tube elongation at 4 hr and had no effect on pollen germination at any time interval tested. Total proteins extracted from control and acid rain stressed pollen grain populations exhibited no differences. The reduction of corn pollen germination in vitro under acid rain stress was mainly due to pollen rupture. The present data indicates the reduction of pollen germination and tube growth under acid rain stress may be a physiological response rather than a genetic response. A simple, nontoxic, and effective method to separate germinated from ungerminated pollen grains has been developed using pollen from corn (Zea mays, L. cv. Pioneer 3747). The separated germinated pollen grains retained viability and continued tube growth when placed in culture medium.

  3. Arabidopsis Microtubule-Destabilizing Protein 25 Functions in Pollen Tube Growth by Severing Actin Filaments[W

    PubMed Central

    Qin, Tao; Liu, Xiaomin; Li, Jiejie; Sun, Jingbo; Song, Leina; Mao, Tonglin

    2014-01-01

    The formation of distinct actin filament arrays in the subapical region of pollen tubes is crucial for pollen tube growth. However, the molecular mechanisms underlying the organization and dynamics of the actin filaments in this region remain to be determined. This study shows that Arabidopsis thaliana MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) has the actin filament–severing activity of an actin binding protein. This protein negatively regulated pollen tube growth by modulating the organization and dynamics of actin filaments in the subapical region of pollen tubes. MDP25 loss of function resulted in enhanced pollen tube elongation and inefficient fertilization. MDP25 bound directly to actin filaments and severed individual actin filaments, in a manner that was dramatically enhanced by Ca2+, in vitro. Analysis of a mutant that bears a point mutation at the Ca2+ binding sites demonstrated that the subcellular localization of MDP25 was determined by cytosolic Ca2+ level in the subapical region of pollen tubes, where MDP25 was disassociated from the plasma membrane and moved into the cytosol. Time-lapse analysis showed that the F-actin-severing frequency significantly decreased and a high density of actin filaments was observed in the subapical region of mdp25-1 pollen tubes. This study reveals a mechanism whereby calcium enhances the actin filament–severing activity of MDP25 in the subapical region of pollen tubes to modulate pollen tube growth. PMID:24424096

  4. P2Y2 nucleotide receptor-mediated extracellular signal-regulated kinases and protein kinase C activation induces the invasion of highly metastatic breast cancer cells.

    PubMed

    Eun, So Young; Ko, Young Shin; Park, Sang Won; Chang, Ki Churl; Kim, Hye Jung

    2015-07-01

    Tumor metastasis is considered the main cause of mortality in cancer patients, thus it is important to investigate the differences between high- and low-metastatic cancer cells. Our previous study showed that the highly metastatic breast cancer cell line MDA-MB-231 released higher levels of ATP and exhibited higher P2Y2R activity compared with the low-metastatic breast cancer cell line MCF-7. In addition, P2Y2R activation by ATP released from MDA-MB-231 cells induced hypoxia-inducible factor-1α expression, lysyl oxidase secretion and collagen crosslinking, generating a receptive microenvironment for pre-metastatic niche formation. Thus, in the present study, we investigated which P2Y2R-related signaling pathways are involved in the invasion of breast cancer cells. The highly metastatic breast cancer cells MDA-MB-231 and SK-BR-3 showed higher invasion than MCF-7 and T47D cells at a basal level, which was abolished through P2Y2R knockdown or in the presence of apyrase, an enzyme that hydrolyzes extracellular nucleotides. MDA-MB-231 cells also showed high levels of mesenchymal markers, such as Snail, Vimentin and N-cadherin, but not the epithelial marker E-cadherin and this expression was inhibited through ATP degradation or P2Y2R knockdown. Moreover, SK-BR-3 and MDA-MB231 cells exhibited higher ERK and PKC phosphorylation levels than T47D and MCF-7 cells and upregulated phospho-ERK and -PKC levels in MDA-MB-231 cells were significantly downregulated by apyrase or P2Y2R knockdown. Specific inhibitors of ERK, PKC and PLC markedly reduced the invasion and levels of mesenchymal marker expression in MDA-MB-231 cells. These results suggest that over-activated ERK and PKC pathways are involved in the P2Y2R-mediated invasion of breast cancer cells.

  5. Identification of a plasma membrane-associated guanine nucleotide exchange factor for ARF6 in chromaffin cells. Possible role in the regulated exocytotic pathway.

    PubMed

    Caumont, A S; Vitale, N; Gensse, M; Galas, M C; Casanova, J E; Bader, M F

    2000-05-26

    ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.

  6. E. coli histidine triad nucleotide binding protein 1 (ecHinT) is a catalytic regulator of D-alanine dehydrogenase (DadA) activity in vivo.

    PubMed

    Bardaweel, Sanaa; Ghosh, Brahma; Chou, Tsui-Fen; Sadowsky, Michael J; Wagner, Carston R

    2011-01-01

    Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both gram-negative and gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli.

  7. Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDN1).

    PubMed Central

    Fukumori, F; Saint, C P

    1997-01-01

    A 9,233-bp HindIII fragment of the aromatic amine catabolic plasmid pTDN1, isolated from a derivative of Pseudomonas putida mt-2 (UCC22), confers the ability to degrade aniline on P. putida KT2442. The fragment encodes six open reading frames which are arranged in the same direction. Their 5' upstream region is part of the direct-repeat sequence of pTDN1. Nucleotide sequence of 1.8 kb of the repeat sequence revealed only a single base pair change compared to the known sequence of IS1071 which is involved in the transposition of the chlorobenzoate genes (C. Nakatsu, J. Ng, R. Singh, N. Straus, and C. Wyndham, Proc. Natl. Acad. Sci. USA 88:8312-8316, 1991). Four open reading frames encode proteins with considerable homology to proteins found in other aromatic-compound degradation pathways. On the basis of sequence similarity, these genes are proposed to encode the large and small subunits of aniline oxygenase (tdnA1 and tdnA2, respectively), a reductase (tdnB), and a LysR-type regulatory gene (tdnR). The putative large subunit has a conserved [2Fe-2S]R Rieske-type ligand center. Two genes, tdnQ and tdnT, which may be involved in amino group transfer, are localized upstream of the putative oxygenase genes. The tdnQ gene product shares about 30% similarity with glutamine synthetases; however, a pUC-based plasmid carrying tdnQ did not support the growth of an Escherichia coli glnA strain in the absence of glutamine. TdnT possesses domains that are conserved among amidotransferases. The tdnQ, tdnA1, tdnA2, tdnB, and tdnR genes are essential for the conversion of aniline to catechol. PMID:8990291

  8. Association between pollen hypersensitivity and edible vegetable allergy: a review.

    PubMed

    Caballero, T; Martín-Esteban, M

    1998-01-01

    Over the last three decades several authors have described the existence of an association between sensitivity to different pollens and sensitivity to diverse edible vegetables. An association between ragweed pollinosis and hypersensitivity to Cucurbitaceae vegetables (e.g., watermelon, melon, cucumber) and banana has been reported. Other authors have found a relationship between birch pollinosis and sensitization to hazelnut, apple, carrot, potato, kiwi and other vegetables. Additionally, several papers have shown the association between mugwort pollinosis and sensitization to celery, carrot, spices, nuts, mustard and Leguminoseae vegetables. Later, some studies showed association between grass pollinosis and sensitization to tomato, potato, green- pea, peanut, watermelon, melon, apple, orange and kiwi. Finally, an association between sensitization to plantain pollen and melon hypersensitivity was also described. The association between pollinosis and edible vegetable sensitization has been explained by the combination of different hypotheses, such as the following: 1) presence of lectins in edible vegetables; 2) existence of IgE to carbohydrates of the glycoproteins (cross-reactive carbohydrate determinants); and, 3) existence of common allergens between pollens and edible vegetables. Up to now three allergens have been identified as responsible for cross-reactivity in these associations: profilin, a 14 kd protein that regulates actin; Bet v 1, the 18 kd birch pollen allergen; and a 60-69 kd allergen. It is important to study in depth these associated sensitizations and the common allergens responsible for them in order to improve diagnostic methods and treatment of these syndromes.

  9. Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice.

    PubMed

    Wu, Jinwen; Shahid, Muhammad Qasim; Chen, Lin; Chen, Zhixiong; Wang, Lan; Liu, Xiangdong; Lu, Yonggen

    2015-12-01

    Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (S(i)S(j)) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility.

  10. Polyploidy Enhances F1 Pollen Sterility Loci Interactions That Increase Meiosis Abnormalities and Pollen Sterility in Autotetraploid Rice1[OPEN

    PubMed Central

    Wu, Jinwen; Chen, Lin; Chen, Zhixiong; Wang, Lan; Lu, Yonggen

    2015-01-01

    Intersubspecific autotetraploid rice (Oryza sativa ssp. indica × japonica) hybrids have greater biological and yield potentials than diploid rice. However, the low fertility of intersubspecific autotetraploid hybrids, which is largely caused by high pollen abortion rates, limits their commercial utility. To decipher the cytological and molecular mechanisms underlying allelic interactions in autotetraploid rice, we developed an autotetraploid rice hybrid that was heterozygous (SiSj) at F1 pollen sterility loci (Sa, Sb, and Sc) using near-isogenic lines. Cytological studies showed that the autotetraploid had higher percentages (>30%) of abnormal chromosome behavior and aberrant meiocytes (>50%) during meiosis than did the diploid rice hybrid control. Analysis of gene expression profiles revealed 1,888 genes that were differentially expressed between the autotetraploid and diploid hybrid lines at the meiotic stage, among which 889 and 999 were up- and down-regulated, respectively. Of the 999 down-regulated genes, 940 were associated with the combined effect of polyploidy and pollen sterility loci interactions (IPE). Gene Ontology enrichment analysis identified a prominent functional gene class consisting of seven genes related to photosystem I (Gene Ontology 0009522). Moreover, 55 meiosis-related or meiosis stage-specific genes were associated with IPE in autotetraploid rice, including Os02g0497500, which encodes a DNA repair-recombination protein, and Os02g0490000, which encodes a component of the ubiquitin-proteasome pathway. These results suggest that polyploidy enhances epistatic interactions between alleles of pollen sterility loci, thereby altering the expression profiles of important meiosis-related or meiosis stage-specific genes and resulting in high pollen sterility. PMID:26511913

  11. Boron Toxicity Causes Multiple Effects on Malus domestica Pollen Tube Growth

    PubMed Central

    Fang, Kefeng; Zhang, Weiwei; Xing, Yu; Zhang, Qing; Yang, Liu; Cao, Qingqin; Qin, Ling

    2016-01-01

    Boron is an important micronutrient for plants. However, boron is also toxic to cells at high concentrations, although the mechanism of this toxicity is not known. This study aimed to evaluate the effect of boron toxicity on Malus domestica pollen tube growth and its possible regulatory pathway. Our results showed that a high concentration of boron inhibited pollen germination and tube growth and led to the morphological abnormality of pollen tubes. Fluorescent labeling coupled with a scanning ion-selective electrode technique detected that boron toxicity could decrease [Ca2+]c and induce the disappearance of the [Ca2+]c gradient, which are critical for pollen tube polar growth. Actin filaments were therefore altered by boron toxicity. Immuno-localization and fluorescence labeling, together with fourier-transform infrared analysis, suggested that boron toxicity influenced the accumulation and distribution of callose, de-esterified pectins, esterified pectins, and arabinogalactan proteins in pollen tubes. All of the above results provide new insights into the regulatory role of boron in pollen tube development. In summary, boron likely plays a structural and regulatory role in relation to [Ca2+]c, actin cytoskeleton and cell wall components and thus regulates Malus domestica pollen germination and tube polar growth. PMID:26955377

  12. A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    PubMed Central

    Romero, David A; Hasan, Ayad H; Lin, Yu-fei; Kime, Louise; Ruiz-Larrabeiti, Olatz; Urem, Mia; Bucca, Giselda; Mamanova, Lira; Laing, Emma E; van Wezel, Gilles P; Smith, Colin P; Kaberdin, Vladimir R; McDowall, Kenneth J

    2014-01-01

    Streptomyces coelicolor is a model for studying bacteria renowned as the foremost source of natural products used clinically. Post-genomic studies have revealed complex patterns of gene expression and links to growth, morphological development and individual genes. However, the underlying regulation remains largely obscure, but undoubtedly involves steps after transcription initiation. Here we identify sites involved in RNA processing and degradation as well as transcription within a nucleotide-resolution map of the transcriptional landscape. This was achieved by combining RNA-sequencing approaches suited to the analysis of GC-rich organisms. Escherichia coli was analysed in parallel to validate the methodology and allow comparison. Previously, sites of RNA processing and degradation had not been mapped on a transcriptome-wide scale for E. coli. Through examples, we show the value of our approach and data sets. This includes the identification of new layers of transcriptional complexity associated with several key regulators of secondary metabolism and morphological development in S. coelicolor and the identification of host-encoded leaderless mRNA and rRNA processing associated with the generation of specialized ribosomes in E. coli. New regulatory small RNAs were identified for both organisms. Overall the results illustrate the diversity in mechanisms used by different bacterial groups to facilitate and regulate gene expression. PMID:25266672

  13. Prokaryotic Nucleotide Excision Repair

    PubMed Central

    Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett

    2013-01-01

    Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation. PMID:23457260

  14. Prokaryotic nucleotide excision repair.

    PubMed

    Kisker, Caroline; Kuper, Jochen; Van Houten, Bennett

    2013-03-01

    Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation.

  15. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate

    PubMed Central

    Carignan, Bailey M.; Brumfield, Kyle D.

    2016-01-01

    ABSTRACT Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple-SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains. IMPORTANCE Cholera, an infectious disease of the small intestine caused by the aquatic bacterium Vibrio cholerae, often results in vomiting and acute watery diarrhea. If left untreated or if the response is too slow, the symptoms can quickly lead to extreme dehydration and ultimately death of the patient. Recent anecdotal evidence of

  16. Single Nucleotide Polymorphisms in Regulator-Encoding Genes Have an Additive Effect on Virulence Gene Expression in a Vibrio cholerae Clinical Isolate.

    PubMed

    Carignan, Bailey M; Brumfield, Kyle D; Son, Mike S

    2016-01-01

    Vibrio cholerae is the etiological agent of the infectious disease cholera, which is characterized by vomiting and severe watery diarrhea. Recently, V. cholerae clinical isolates have demonstrated increased virulence capabilities, causing more severe symptoms with a much higher rate of disease progression than previously observed. We have identified single nucleotide polymorphisms (SNPs) in four virulence-regulatory genes (hapR, hns, luxO, and vieA) of a hypervirulent V. cholerae clinical isolate, MQ1795. Herein, all SNPs and SNP combinations of interest were introduced into the prototypical El Tor reference strain N16961, and the effects on the production of numerous virulence-related factors, including cholera toxin (CT), the toxin-coregulated pilus (TCP), and ToxT, were analyzed. Our data show that triple-SNP (hapR hns luxO and hns luxO vieA) and quadruple-SNP combinations produced the greatest increases in CT, TCP, and ToxT production. The hns and hns luxO SNP combinations were sufficient for increased TCP and ToxT production. Notably, the hns luxO vieA triple-SNP combination strain produced TCP and ToxT levels similar to those of MQ1795. Certain SNP combinations (hapR and hapR vieA) had the opposite effect on CT, TCP, and ToxT expression. Interestingly, the hns vieA double-SNP combination strain increased TCP production while decreasing CT production. Our findings suggest that SNPs identified in the four regulatory genes, in various combinations, are associated with increased virulence capabilities observed in V. cholerae clinical isolates. These studies provide insight into the evolution of highly virulent strains. IMPORTANCE Cholera, an infectious disease of the small intestine caused by the aquatic bacterium Vibrio cholerae, often results in vomiting and acute watery diarrhea. If left untreated or if the response is too slow, the symptoms can quickly lead to extreme dehydration and ultimately death of the patient. Recent anecdotal evidence of cholera

  17. Changes in NMDA receptor-induced cyclic nucleotide synthesis regulate the age-dependent increase in PDE4A expression in primary cortical cultures

    PubMed Central

    Hajjhussein, Hassan; Suvarna, Neesha U.; Gremillion, Carmen; Judson Chandler, L.; O’Donnell, James M.

    2007-01-01

    NMDA receptor-induced cAMP and cGMP are selectively hydrolyzed by PDE4 and PDE2, respectively, in rat primary cerebral cortical and hippocampal cultures. Because cAMP levels regulate the expression of PDE4 in rat primary cortical cultures, we examined the manner in which NMDA receptor activity regulates the age-dependent increase in the expression of PDE4A observed in vivo and in vitro. Inhibiting the activity of NR2B subunit with ifenprodil blocked NMDA receptor-induced cGMP synthesis and increased NMDA receptor-induced cAMP levels in a manner that reduced PDE4 activity. Therefore, NR1/NR2B receptor-induced cGMP signaling is involved in an acute cross-talk regulation of NR1/NR2A receptor-induced cAMP levels, mediated by PDE4. Chronic inhibition of NMDA receptor activity with MK-801 reduced PDE4A1 and PDE4A5 expression and activity in a time-dependent manner; this effect was reversed by adding the PKA activator dbr-cAMP. Inhibiting GABA receptors with bicuculline increased NMDA receptor-induced cAMP synthesis and PDE4A expression in cultures treated between DIV 16 and DIV 21 but not in cultures treated between DIV 8 and DIV 13. This effect was due to a high tone of NMDA receptor-induced cGMP in younger cultures, which negatively regulated the expression of PDE4A by a PKG-mediated process. The present results are consistent with behavioral data showing that both PDE4 and PDE2 are involved in NMDA receptor-mediated memory processes. PMID:17407767

  18. Tree pollen spectra and pollen allergy risk in the Osijek-Baranja County.

    PubMed

    Sikora, Magdalena; Valek, Marina; Šušić, Zdenka; Santo, Vera; Brdarić, Dario

    2013-01-01

    The forests of north-eastern Croatia, as well as various plants and trees in the parks and streets of the Osijek-Baranja County, produce large amounts of pollen during the pollen season, which can cause allergy symptoms in pollen sensitive individuals. The aim of this study was to determine the most frequent types of pollen in this area and estimate possible health risks, especially the risk of allergy. In 2009 and 2010, the staff of the Health Ecology Department of the Osijek Public Health Institute monitored tree pollen concentrations in four cities from the Osijek - Baranja County (Osijek, Našice, Đakovo and Beli Manastir) using a Burkard volumetric instrument. The results were affected by weather conditions. Windy and sunny days facilitated the transfer of pollen, whereas during rainy days, the concentration of pollen grains decreased. High pollen concentrations of Cupressaceae/Taxaceae, Betulaceae, Salicaceae and Aceraceae could be the cause for symptoms of pollen allergy. In 2009, conifers, birch and poplar pollen were dominant at all monitoring stations with 5000 pollen grains (PG), 3188 PG and 3113 PG respectively. The highest number of pollen grains was recorded at measuring site Osijek. The variations in airborne pollen concentration between pollen seasons were recorded at all monitoring stations. The most obvious variations were recorded at measuring site Osijek. The usual pollination period lasts two to three months, which means that most pollen grains remain present from February to early June. However, the Cupressaceae / Taxaceae pollination periods last the longest and their pollen grains remain present until the end of summer. The risk of allergy was determined at four monitored measuring stations and the obtained data confirmed that the largest number of days with a high health risk was at the Đakovo measuring station for a species of birch. The research information aims to help allergologists and individuals allergic to plant pollen develop

  19. Towards a "crime pollen calendar" - pollen analysis on corpses throughout one year.

    PubMed

    Montali, Elisa; Mercuri, Anna Maria; Trevisan Grandi, Giuliana; Accorsi, Carla Alberta

    2006-11-22

    A palynological study was carried out on 28 corpses brought in one year (June 2003-May 2004) to the morgue of the Institute of Legal Medicine of Parma (Northern Italy). This preliminary research focuses on the date of death, which was known for all corpses examined. Pollen sampling and analyses were made with the first aim of comparing the pollen grains found on corpses with those diffused in the atmosphere in the region in the same season as the known date of death. Eyebrows, hair-line near the forehead, facial skin and nasal cavities were sampled. Most of the corpses had trapped pollen grains, with the exception of two December corpses. All pollen grains were found with cytoplasm and in a good state of preservation. In this way, a series of reference data was collected for the area where the deaths occurred, and we examined whether pollen grains on corpses could be an index of the season of death. To verify this hypothesis, the pollen analyses were compared with data reported in the airborne pollen calendars of Parma and the region around. Pollen calendars record pollen types and their concentrations in the air, month by month. The quantity of pollen recorded on corpses did not prove to be directly related to the quantity of pollen in the air. But qualitatively, many pollen types which are seasonal markers were found on corpses. Main corpse/air discrepancies were also observed due to the great influence that the local environmental conditions of the death scene have in determining the pollen trapped by a corpse. Qualitative plus quantitative pollen data from corpses appeared helpful in indicating the season of death. A preliminary sketch of a "crime pollen calendar" in a synthetic graphic form was made by grouping the corpse pollen records into three main seasons: A, winter/spring; B, spring/summer; C, summer/autumn. Trends match the general seasonal trend of pollen types in the air.

  20. Pollen-Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen

    PubMed Central

    Obersteiner, Andrea; Gilles, Stefanie; Frank, Ulrike; Beck, Isabelle; Häring, Franziska; Ernst, Dietrich; Rothballer, Michael; Hartmann, Anton; Traidl-Hoffmann, Claudia; Schmid, Michael

    2016-01-01

    Pollen allergies have been rapidly increasing over the last decades. Many allergenic proteins and non-allergenic adjuvant compounds of pollen are involved in the plant defense against environmental or microbial stress. The first aim of this study was to analyze and compare the colonizing microbes on allergenic pollen. The second aim was to investigate detectable correlations between pollen microbiota and parameters of air pollution or pollen allergenicity. To reach these aims, bacterial and fungal DNA was isolated from pollen samples of timothy grass (Phleum pratense, n = 20) and birch trees (Betula pendula, n = 55). With this isolated DNA, a terminal restriction fragment length polymorphism analysis was performed. One result was that the microbial diversity on birch tree and timothy grass pollen samples (Shannon/Simpson diversity indices) was partly significantly correlated to allergenicity parameters (Bet v 1/Phl p 5, pollen-associated lipid mediators). Furthermore, the microbial diversity on birch pollen samples was correlated to on-site air pollution (nitrogen dioxide (NO2), ammonia (NH3), and ozone (O3)). What is more, a significant negative correlation was observed between the microbial diversity on birch pollen and the measured NO2 concentrations on the corresponding trees. Our results showed that the microbial composition of pollen was correlated to environmental exposure parameters alongside with a differential expression of allergen and pollen-associated lipid mediators. This might translate into altered allergenicity of pollen due to environmental and microbial stress. PMID:26910418

  1. Pollen development in Annona cherimola Mill. (Annonaceae). Implications for the evolution of aggregated pollen

    PubMed Central

    Lora, Jorge; Testillano, Pilar S; Risueño, Maria C; Hormaza, Jose I; Herrero, Maria

    2009-01-01

    Background In most flowering plants, pollen is dispersed as monads. However, aggregated pollen shedding in groups of four or more pollen grains has arisen independently several times during angiosperm evolution. The reasons behind this phenomenon are largely unknown. In this study, we followed pollen development in Annona cherimola, a basal angiosperm species that releases pollen in groups of four, to investigate how pollen ontogeny may explain the rise and establishment of this character. We followed pollen development using immunolocalization and cytochemical characterization of changes occurring from anther differentiation to pollen dehiscence. Results Our results show that, following tetrad formation, a delay in the dissolution of the pollen mother cell wall and tapetal chamber is a key event that holds the four microspores together in a confined tapetal chamber, allowing them to rotate and then bind through the aperture sites through small pectin bridges, followed by joint sporopollenin deposition. Conclusion Pollen grouping could be the result of relatively minor ontogenetic changes beneficial for pollen transfer or/and protection from desiccation. Comparison of these events with those recorded in the recent pollen developmental mutants in Arabidopsis indicates that several failures during tetrad dissolution may convert to a common recurring phenotype that has evolved independently several times, whenever this grouping conferred advantages for pollen transfer. PMID:19874617

  2. Pollen-Associated Microbiome Correlates with Pollution Parameters and the Allergenicity of Pollen.

    PubMed

    Obersteiner, Andrea; Gilles, Stefanie; Frank, Ulrike; Beck, Isabelle; Häring, Franziska; Ernst, Dietrich; Rothballer, Michael; Hartmann, Anton; Traidl-Hoffmann, Claudia; Schmid, Michael

    2016-01-01

    Pollen allergies have been rapidly increasing over the last decades. Many allergenic proteins and non-allergenic adjuvant compounds of pollen are involved in the plant defense against environmental or microbial stress. The first aim of this study was to analyze and compare the colonizing microbes on allergenic pollen. The second aim was to investigate detectable correlations between pollen microbiota and parameters of air pollution or pollen allergenicity. To reach these aims, bacterial and fungal DNA was isolated from pollen samples of timothy grass (Phleum pratense, n = 20) and birch trees (Betula pendula, n = 55). With this isolated DNA, a terminal restriction fragment length polymorphism analysis was performed. One result was that the microbial diversity on birch tree and timothy grass pollen samples (Shannon/Simpson diversity indices) was partly significantly correlated to allergenicity parameters (Bet v 1/Phl p 5, pollen-associated lipid mediators). Furthermore, the microbial diversity on birch pollen samples was correlated to on-site air pollution (nitrogen dioxide (NO2), ammonia (NH3), and ozone (O3)). What is more, a significant negative correlation was observed between the microbial diversity on birch pollen and the measured NO2 concentrations on the corresponding trees. Our results showed that the microbial composition of pollen was correlated to environmental exposure parameters alongside with a differential expression of allergen and pollen-associated lipid mediators. This might translate into altered allergenicity of pollen due to environmental and microbial stress.

  3. Pollen tube energetics: respiration, fermentation and the race to the ovule.

    PubMed

    Rounds, Caleb M; Winship, Lawrence J; Hepler, Peter K

    2011-01-01

    Pollen tubes grow by transferring chemical energy from stored cellular starch and newly assimilated sugars into ATP. This drives myriad processes essential for cell elongation, directly or through the creation of ion gradients. Respiration plays a central role in generating and regulating this energy flow and thus in the success of plant reproduction. Pollen tubes are easily grown in vitro and have become an excellent model for investigating the contributions of respiration to plant cellular growth and morphogenesis at the molecular, biochemical and physiological levels. In recent decades, pollen tube research has become increasingly focused on the molecular mechanisms involved in cellular processes. Yet, effective growth and development requires an intact, integrated set of cellular processes, all supplied with a constant flow of energy. Here we bring together information from the current and historical literature concerning respiration, fermentation and mitochondrial physiology in pollen tubes, and assess the significance of more recent molecular and genetic investigations in a physiological context. The rapid growth of the pollen tube down the style has led to the evolution of high rates of pollen tube respiration. Respiration rates in lily predict a total energy turnover of 40-50 fmol ATP s(-1) per pollen grain. Within this context we examine the energetic requirements of cell wall synthesis, osmoregulation, actin dynamics and cyclosis. At present, we can only estimate the amount of energy required, because data from growing pollen tubes are not available. In addition to respiration, we discuss fermentation and mitochondrial localization. We argue that the molecular pathways need to be examined within the physiological context to understand better the mechanisms that control tip growth in pollen tubes.

  4. Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides

    PubMed Central

    Zhang, Xiao-Ling; Zhang, Jin; Guo, Ying-Hua; Sun, Pei; Jia, Hui-Xia; Fan, Wei; Lu, Meng-Zhu; Hu, Jian-Jun

    2016-01-01

    Ploidy affects plant growth vigor and cell size, but the relative effects of pollen fertility and allergenicity between triploid and diploid have not been systematically examined. Here we performed comparative analyses of fertility, proteome, and abundances of putative allergenic proteins of pollen in triploid poplar ‘ZhongHuai1’ (‘ZH1’, triploid) and ‘ZhongHuai2’ (‘ZH2’, diploid) generated from the same parents. The mature pollen was sterile in triploid poplar ‘ZH1’. By applying two-dimensional gel electrophoresis (2-DE), a total of 72 differentially expressed protein spots (DEPs) were detected in triploid poplar pollen. Among them, 24 upregulated and 43 downregulated proteins were identified in triploid poplar pollen using matrix-assisted laser desorption/ionisation coupled with time of-flight tandem mass spectrometer analysis (MALDI-TOF/TOF MS/MS). The main functions of these DEPs were related with “S-adenosylmethionine metabolism”, “actin cytoskeleton organization”, or “translational elongation”. The infertility of triploid poplar pollen might be related to its abnormal cytoskeletal system. In addition, the abundances of previously identified 28 putative allergenic proteins were compared among three poplar varieties (‘ZH1’, ‘ZH2’, and ‘2KEN8‘). Most putative allergenic proteins were downregulated in triploid poplar pollen. This work provides an insight into understanding the protein regulation mechanism of pollen infertility and low allergenicity in triploid poplar, and gives a clue to improving poplar polyploidy breeding and decreasing the pollen allergenicity. PMID:27598155

  5. [Identification of cattail pollen (puhuang), pine pollen (songhuafen) and its adulterants by ITS2 sequence].

    PubMed

    Ma, Xiao-Xi; Sun, Wei; Ren, Wei-Chao; Xiang, Li; Zhao, Bo; Zhang, Ya-Qin; Song, Ming; Mu, Ze-Jing; Chen, Shi-Lin

    2014-06-01

    DNA barcoding method was conducted for the authentication of pollen materials due to difficulty of discriminating pollen materials bearing morphological similarity. In this study, a specific focus was to identify cattail pollen (Puhuang) and pine pollen (Songhuafen) samples from their adulterants which are frequently mixed-together. Regions of the internal transcribed spacer (ITS2) from 60 samples were sequenced, and new primers for cattail pollen were designed according to the sequence information. The results from the NJ trees showed that the species of pine pollen, Puhuang and their adulterants can be classified as obvious monophyly. Therefore, we propose to adapt DNA barcoding methodology to accurately distinguish cattail pollen, pine pollen and their adulterant materials. It is a great help for drug regulatory agency to supervise the quality of medicinal materials.

  6. Pollen Raman spectra database: application to the identification of airborne pollen.

    PubMed

    Guedes, A; Ribeiro, H; Fernández-González, M; Aira, M J; Abreu, I

    2014-02-01

    Raman microspectroscopy allows a non-destructive identification of airborne particles. However, the identification of particles such as pollen is hindered by the absence of a spectral library. Although reference spectra of pollen have been published before, they have always been limited to a certain number of species. In this work, Raman spectra of 34 pollen types are presented and were used to build a pollen spectra primary library. Afterward, the applicability of this database for detecting and identifying pollen in airborne samples was tested. Airborne pollen samples collected during April, May and August were compared with blank pollen spectra by means of Hit Quality Index. Although a much larger library would be required, our results showed that all first hits correspond to the same blank pollen species of the questioned sample from the air. This possibility is an innovative idea and a promising line of investigation for future RAMAN technology development in the area of aerobiology.

  7. Disentangling the effects of feedback structure and climate on Poaceae annual airborne pollen fluctuations and the possible consequences of climate change.

    PubMed

    García de León, David; García-Mozo, Herminia; Galán, Carmen; Alcázar, Purificación; Lima, Mauricio; González-Andújar, José L

    2015-10-15

    Pollen allergies are the most common form of respiratory allergic disease in Europe. Most studies have emphasized the role of environmental processes, as the drivers of airborne pollen fluctuations, implicitly considering pollen production as a random walk. This work shows that internal self-regulating processes of the plants (negative feedback) should be included in pollen dynamic systems in order to give a better explanation of the observed pollen temporal patterns. This article proposes a novel methodological approach based on dynamic systems to investigate the interaction between feedback structure of plant populations and climate in shaping long-term airborne Poaceae pollen fluctuations and to quantify the effects of climate change on future airborne pollen concentrations. Long-term historical airborne Poaceae pollen data (30 years) from Cordoba city (Southern Spain) were analyzed. A set of models, combining feedback structure, temperature and actual evapotranspiration effects on airborne Poaceae pollen were built and compared, using a model selection approach. Our results highlight the importance of first-order negative feedback and mean annual maximum temperature in driving airborne Poaceae pollen dynamics. The best model was used to predict the effects of climate change under two standardized scenarios representing contrasting temporal patterns of economic development and CO2 emissions. Our results predict an increase in pollen levels in southern Spain by 2070 ranging from 28.5% to 44.3%. The findings from this study provide a greater understanding of airborne pollen dynamics and how climate change might impact the future evolution of airborne Poaceae pollen concentrations and thus the future evolution of related pollen allergies. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. The adaptor protein 3BP2 associates with VAV guanine nucleotide exchange factors to regulate NFAT activation by the B-cell antigen receptor.

    PubMed

    Foucault, Isabelle; Le Bras, Séverine; Charvet, Céline; Moon, Chéol; Altman, Amnon; Deckert, Marcel

    2005-02-01

    Engagement of the B-cell antigen receptor (BCR) activates kinases of the Src and Syk families and signaling complexes assembled by adaptor proteins, which dictate B-cell fate and function. The adaptor 3BP2/SH3BP2, an Abl Src homology domain 3 (SH3)-binding and Syk-kinases interacting protein, exhibits positive regulatory roles in T, natural killer (NK), and basophilic cells. However, its involvement in BCR signaling is completely unknown. Here we show that 3BP2 is tyrosine phosphorylated following BCR aggregation on B lymphoma cells, and that 3BP2 is a substrate for Syk and Fyn, but not Btk. To further explore the function of 3BP2 in B cells, we screened a yeast 2-hybrid B-lymphocyte library and found 3BP2 as a binding partner of Vav proteins. The interaction between 3BP2 and Vav proteins involved both constitutive and inducible mechanisms. 3BP2 also interacted with other components of the BCR signaling pathway, including Syk and phospholipase C gamma (PLC-gamma). Furthermore, overexpression and RNAi blocking experiments showed that 3BP2 regulated BCR-mediated activation of nuclear factor of activated T cells (NFATs). Finally, evidence was provided that 3BP2 functionally cooperates with Vav proteins and Rho GTPases to activate NFATs. Our results show that 3BP2 may regulate BCR-mediated gene activation through Vav proteins.

  9. Potassium flux in the pollen tubes was essential in plant sexual reproduction.

    PubMed

    Wu, Ju-You; Jin, Cong; Zhang, Shao-Ling

    2011-06-01

    Potassium channels are controlling K (+) transport across plasma membrane and thus playing a central role in all aspects of osmolarity as well as numerous other functions in plants including in sexual reproduction. We have used whole-cell and single-channel patch-clamp recording techniques investigated the regulation of intracellular free Ca ( 2+) -activated outward K (+) channels in Pyrus pyrifolia pollen tube protoplasts. We have also showed the channels could be inhibited by heme and activated carbon monoxide (CO). In the presence of oxygen and NADPH, hemoxygenases catalyzes heme degradation, producing biliverdin, iron and CO. Considered the oxygen concentration approaching zero in the ovary, the heme will inhibit the K (+) outward flux from the intracellular of pollen tube, increasing the pollen tubes osmolarity, inducing pollen tube burst. Here we discuss the putative role of K (+) channels in plant sexual reproduction.

  10. Variable Expression of Pathogenesis-Related Protein Allergen in Mountain Cedar (Juniperus ashei) Pollen1

    PubMed Central

    Midoro-Horiuti, Terumi; Goldblum, Randall M.; Kurosky, Alexander; Wood, Thomas G.; Brooks, Edward G.

    2009-01-01

    Allergic diseases have been increasing in industrialized countries. The environment is thought to have both direct and indirect modulatory effects on disease pathogenesis, including alterating on the allergenicity of pollens. Certain plant proteins known as pathogenesis-related proteins appear to be up-regulated by certain environmental conditions, including pollutants, and some have emerged as important allergens. Thus, the prospect of environmentally regulated expression of plant-derived allergens becomes yet another potential environmental influence on allergic disease. We have identified a novel pathogenesis-related protein allergen, Jun a 3, from mountain cedar (Juniperus ashei) pollen. The serum IgE from patients with hypersensitivity to either mountain cedar or Japanese cedar were shown to bind to native and recombinant Jun a 3 in Western blot analysis and ELISA. Jun a 3 is homologous to members of the thaumatin-like pathogenesis-related (PR-5) plant protein family. The amounts of Jun a 3 extracted from mountain cedar pollen varied up to 5-fold in lots of pollen collected from the same region in different years and between different regions during the same year. Thus, Jun a 3 may contribute not only to the overall allergenicity of mountain cedar pollen, but variable levels of Jun a 3 may alter the allergenic potency of pollens produced under different environmental conditions. PMID:10657673

  11. Heterospecific pollen deposition in Delphinium barbeyi: linking stigmatic pollen loads to reproductive output in the field

    PubMed Central

    Briggs, Heather M.; Anderson, Lucy M.; Atalla, Laila M.; Delva, André M.; Dobbs, Emily K.; Brosi, Berry J.

    2016-01-01

    Background and Aims Most pollinators are generalists and therefore are likely to transfer heterospecific pollen among co-flowering plants. Most work on the impacts of heterospecific pollen deposition on plant fecundity has utilized hand-pollination experiments in greenhouse settings, and we continue to know very little about the reproductive effects of heterospecific pollen in field settings. Methods We explored how patterns of naturally deposited heterospecific pollen relate to the reproductive output of Delphinium barbeyi, a common subalpine perennial herb in the Rocky Mountains (USA). We assessed a wide range of naturally occurring heterospecific pollen proportions and pollen load sizes, and linked stigmatic pollen deposition directly to seed set in individual carpels in the field. Key Results We found that heterospecific pollen deposition in D. barbeyi is common, but typically found at low levels across stigmas collected in our sites. Neither conspecific nor heterospecific pollen deposition was related to carpel abortion. By contrast, we saw a significant positive relationship between conspecific pollen amount and viable seed production, as well as a significant negative interaction between the effects of conspecific pollen and heterospecific pollen amount, whereby the effect of conspecific pollen on viable seed production became weaker with greater heterospecific deposition on stigmas. Conclusions To our knowledge, this is the first demonstration of a relationship between heterospecific pollen and seed production in a field setting. In addition, it is the first report of an interaction between conspecific and heterospecific pollen quantities on seed production. These findings, taken with the results from other studies, suggest that greenhouse hand-pollination studies and field studies should be more tightly integrated in future work to better understand how heterospecific pollen transfer can be detrimental for plant reproduction. PMID:26658101

  12. A single nucleotide polymorphism in the 3′-UTR of STAT3 regulates its expression and reduces risk of pancreatic cancer in a Chinese population

    PubMed Central

    Lou, Jiao; Ke, Juntao; Zhang, Yi; Li, Jiaoyuan; Gong, Yajie; Yang, Yang; Tian, Jianbo; Peng, Xiating; Zou, Danyi; Zhong, Rong; Gong, Jing; Chang, Jiang; Li, Lu; Miao, Xiaoping

    2016-01-01

    Pancreatic cancer (PC) is one of the deadliest solid malignancies carrying a gloomy 5-year survival rate less than 5%. The signal transducer and activator of transcription 3 (STAT3) is a common transcriptional regulator, whose aberrant expression has been widely found in human cancers, including PC. Our current study aimed to illustrate the roles of common variants, in the three prime untranslated region (3′UTR) of STAT3, in modifying the risk of PC through two-stage case-control studies integrating biological experiments. We first explored the associations between two common variants (rs1053004 and rs1053005) and PC risk in 774 PC cases and 777 controls. Only rs1053004 T > C showed a significant association with a reduced risk of PC with an odds ratio (OR) and 95% confidence interval (CI) of 0.85 (0.74–0.98). Then we attempted to validate the association in another 940 cases and 1398 controls, and the significant association persisted with OR (95%CI) of 0.86 (0.76–0.97). Dual luciferase reporter gene assays indicated that C allele conferred a higher expression of STAT3 in three PC cell lines including Panc-1 (P = 3.0 × 10−3), BxPC-3 (P = 6.7 × 10−5) and SW1990 (P = 4.0 × 10−3). In conclusion, the current study provided evidence that rs1053004 T > C in 3′UTR of STAT3 may decrease the risk of PC through up-regulating the gene expression. PMID:27577070

  13. Self-incompatibility-induced programmed cell death in field poppy pollen involves dramatic acidification of the incompatible pollen tube cytosol.

    PubMed

    Wilkins, Katie A; Bosch, Maurice; Haque, Tamanna; Teng, Nianjun; Poulter, Natalie S; Franklin-Tong, Vernonica E

    2015-03-01

    Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH]cyt of the pollen tubes in vivo, we show that [pH]cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH]cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and Actin-Depolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH]cyt and its effect on several components in the context of SI-induced events and PCD.

  14. Leucine Zipper Down-regulated in Cancer-1 (LDOC1) interacts with Guanine nucleotide binding protein-like 3-like (GNL3L) to modulate Nuclear Factor-kappa B (NF-κB) signaling during cell proliferation.

    PubMed

    Thoompumkal, Indu Jose; Rehna, Krishnan; Anbarasu, Kumaraswamy; Mahalingam, Sundarasamy

    2016-12-01

    Guanine nucleotide binding protein-like 3-like (GNL3L) is an evolutionarily conserved putative nucleolar GTPase belonging to the HSR1-MMR1 family. In the present study, using protein-protein interaction assays, we show that Leucine Zipper Down-regulated in Cancer-1 (LDOC1) is a novel interacting partner of GNL3L. Furthermore, our results reveal that ectopic expression of LDOC1 destabilizes endogenous GNL3L levels and down modulates GNL3L-induced cell proliferation, in contrast, the knockdown of LDOC1 potentiates cell proliferation upon GNL3L expression. Interestingly, GNL3L upregulates NF-κB dependent transcriptional activity by modulating the expression of NF-κB subunit p65, which is reversed upon co-expression of LDOC1 with GNL3L. GNL3L also potentiates TNF-α mediated NF-κB activity. In addition, anti-apoptotic function of GNL3L is impaired upon p65 knockdown, suggesting its critical role in GNL3L mediated cell proliferation/survival. An inverse correlation of GNL3L and LDOC1 expression profiles in various tumor tissues from BioXpress database indicate their critical role in cancer. Collectively, our data provides evidence that GNL3L-LDOC1 interplay regulates cell proliferation through the modulation of NF-κB pathway during tumorigenesis.

  15. Cross-reactivity to olive tree pollen and orchard grass pollen in patients with pollinosis.

    PubMed

    Miyahara, S; Nakada, M; Nishizaki, K; Kawarai, Y; Nishioka, K; Hino, H

    1997-06-01

    We studied 92 patients with allergic rhinitis in Syodoshima, Japan, during the pollen season between April and June to evaluate the cross-reactivity to different antigens, including pollen from the olive tree (Olea europaea) and orchard grass (Dactylis glomerata). Olive tree pollen was present in the atmosphere for 23 days, from May 19 to June 12, 1994. Specific IgE antibodies for olive tree pollen antigen were present in 21 (26.9%) of the 78 patients with allergic rhinitis. Nine (24.3%) of the 37 patients with allergic rhinitis exhibited positive skin reactivity to an extract of olive tree pollen. Fifteen (88.2%) of the 17 patients who had IgE reactivity in their sera to olive tree pollen antigen demonstrated allergic reactions to an extract of olive tree pollen. Specific IgE antibodies for orchard grass pollen antigen were present in 43 (48.3%) of the 89 patients with allergic rhinitis and 20 (95.2%) of the 21 patients who had IgE reactivity in their sera to olive tree pollen antigen. The inhibition test using the CAP System revealed that the reactivity of the IgE antibody specific for olive tree pollen antigen was inhibited dose-dependently by an extract of orchard grass pollen. These findings show that there is a reaction in some patients with grass (Gramineae) pollinosis that might be induced by olive tree pollen.

  16. In Vitro Pollen Viability and Pollen Germination in Cherry Laurel (Prunus laurocerasus L.)

    PubMed Central

    Sulusoglu, Melekber; Cavusoglu, Aysun

    2014-01-01

    Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability and pollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.). Two pollen viability tests, TTC (2,3,5-triphenyl tetrazolium chloride) and IKI (iodine potassium iodide), were used. Pollen traits of genotypes were studied using an in vitro medium containing 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step, the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rates were different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation (r2 = 0.0614 and r2 = 0.0015, resp.). Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viability estimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germination rates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose and the results showed that pollen germination rates increased after 6 hours of being placed in culture media. PMID:25405230

  17. Seed set, pollen morphology and pollen surface composition response to heat stress in field pea.

    PubMed

    Jiang, Yunfei; Lahlali, Rachid; Karunakaran, Chithra; Kumar, Saroj; Davis, Arthur R; Bueckert, Rosalind A

    2015-11-01

    Pea (Pisum sativum L.) is a major legume crop grown in a semi-arid climate in Western Canada, where heat stress affects pollination, seed set and yield. Seed set and pod growth characteristics, along with in vitro percentage pollen germination, pollen tube growth and pollen surface composition, were measured in two pea cultivars (CDC Golden and CDC Sage) subjected to five maximum temperature regimes ranging from 24 to 36 °C. Heat stress reduced percentage pollen germination, pollen tube length, pod length, seed number per pod, and the seed-ovule ratio. Percentage pollen germination of CDC Sage was greater than CDC Golden at 36 °C. No visible morphological differences in pollen grains or the pollen surface were observed between the heat and control-treated pea. However, pollen wall (intine) thickness increased due to heat stress. Mid-infrared attenuated total reflectance (MIR-ATR) spectra revealed that the chemical composition (lipid, proteins and carbohydrates) of each cultivar's pollen grains responded differently to heat stress. The lipid region of the pollen coat and exine of CDC Sage was more stable compared with CDC Golden at 36 °C. Secondary derivatives of ATR spectra indicated the presence of two lipid types, with different amounts present in pollen grains from each cultivar. © 2015 John Wiley & Sons Ltd.

  18. Some carbohydrates found in pollen and pollen substitutes are toxic to honey bees.

    PubMed

    Barker, R J

    1977-10-01

    Carbohydrates in some pollen substitutes (galactose, lactose, raffinose, stachyose, glucuronic acid, galacturonic acid, polygalacturonic acid, and pectin) were toxic to caged adult Apis mellifera L. These toxins can be diluted to safe levels by sucrose. Collected nectar apparently dilutes the toxic sugars in pollen thus permitting assimilation of essential nutrients from pollen.

  19. In vitro pollen viability and pollen germination in cherry laurel (Prunus laurocerasus L.).

    PubMed

    Sulusoglu, Melekber; Cavusoglu, Aysun

    2014-01-01

    Pollen quality is important for growers and breeders. This study was carried out to determine in vitro pollen viability and pollen germination in seven genotypes of cherry laurel (Prunus laurocerasus L.). Two pollen viability tests, TTC (2,3,5-triphenyl tetrazolium chloride) and IKI (iodine potassium iodide), were used. Pollen traits of genotypes were studied using an in vitro medium containing 0%, 5%, 10%, 15%, and 20% sucrose to determine the best sucrose concentrations for germination. In the second step, the germinated pollen was counted 1, 4, 6, 10, 12, 24, and 48 hours later until there was no further germination. The viability rates were different according to genotypes and tests used. The IKI and TTC staining tests and pollen germination had low correlation (r(2) = 0.0614 and r(2) = 0.0015, resp.). Painted pollen rate was higher and pollen was well-stained with IKI test and pollen viability estimated with TTC staining test was better than that estimated with the IKI staining test. 15% sucrose gave the best germination rates in most of the genotypes. Pollen germination rates were recorded periodically from one hour to 48 hours in 15% sucrose and the results showed that pollen germination rates increased after 6 hours of being placed in culture media.

  20. [A monoclonal antibody against ragweed pollen cross-reacting with yellow dock pollen].

    PubMed

    Shen, H D; Chang, L Y; Gong, Y J; Chang, H N; Han, S H

    1985-11-01

    Using monoclonal antibodies with different specificity against the major allergenic components of ragweed pollen, we analyzed their cross-reactivity with two tree pollens, two grass pollens and five other weed pollens which are common in Taiwan by the immunoblot method. It was found that besides reacting with AgE and AgK of the ragweed pollen, the monoclonal antibody 48-5 also reacted with antigens of yellow dock pollen with molecular weights of 40K, 38K, 24K, and 21K. In a preliminary study, sera of two patients containing IgE antibodies to ragweed pollen antigens also reacted to the 40K component of the yellow dock pollen. Furthermore, from the results of allergenic skin testings on 109 patients with bronchial asthma, we found that of 22 patients who had a positive reaction to a crude extract of ragweed pollen, 18(81.8%) also reacted to the crude extract of yellow dock pollen. In conclusion, our results suggest that there exists a common allergenic determinant between pollens of ragweed and yellow dock. It may play an important role in the expression of the sensitivity of patients to these two kinds of pollens.

  1. Transcriptional Evidence for Inferred Pattern of Pollen Tube-Stigma Metabolic Coupling during Pollination

    PubMed Central

    Wang, Fang; Dong, YuXiu; Li, XingGuo; Zhang, Xian Sheng

    2014-01-01

    It is difficult to derive all qualitative proteomic and metabolomic experimental data in male (pollen tube) and female (pistil) reproductive tissues during pollination because of the limited sensitivity of current technology. In this study, genome-scale enzyme correlation network models for plants (Arabidopsis/maize) were constructed by analyzing the enzymes and metabolic routes from a global perspective. Then, we developed a data-driven computational pipeline using the “guilt by association” principle to analyze the transcriptional coexpression profiles of enzymatic genes in the consecutive steps for metabolic routes in the fast-growing pollen tube and stigma during pollination. The analysis identified an inferred pattern of pollen tube-stigma ethanol coupling. When the pollen tube elongates in the transmitting tissue (TT) of the pistil, this elongation triggers the mobilization of energy from glycolysis in the TT cells of the pistil. Energy-rich metabolites (ethanol) are secreted that can be taken up by the pollen tube, where these metabolites are incorporated into the pollen tube's tricarboxylic acid (TCA) cycle, which leads to enhanced ATP production for facilitating pollen tube growth. In addition, our analysis also provided evidence for the cooperation of kaempferol, dTDP-alpha-L-rhamnose and cell-wall-related proteins; phosphatidic-acid-mediated Ca2+ oscillations and cytoskeleton; and glutamate degradation IV for γ-aminobutyric acid (GABA) signaling activation in Arabidopsis and maize stigmas to provide the signals and materials required for pollen tube tip growth. In particular, the “guilt by association” computational pipeline and the genome-scale enzyme correlation network models (GECN) developed in this study was initiated with experimental “omics” data, followed by data analysis and data integration to determine correlations, and could provide a new platform to assist inachieving a deeper understanding of the co-regulation and inter-regulation

  2. Personal pollen exposure compared to stationary measurements.

    PubMed

    Riediker, M; Keller, S; Wüthrich, B; Koller, T; Monn, C

    2000-01-01

    The aim of this study was to examine to what extent stationary outdoor pollen measurements are representative for estimating personal exposure to pollen. Ten subjects were studied during a total of 36 days in spring and summer Pollen was sampled using personal SKC total dust samplers and stationary Burkard pollen traps. The personal activity pattern was recorded quarter-hourly as well as the time spent outdoors. As a reference, SKC and Burkard samplers were run stationary and in parallel. Stationary comparison of the samplers showed good correlation (r = 0.981, p <0.001). However, the SKC sampler collected systematically about four times less pollen than the Burkard sampler. Taking into account the systematic difference between the sampling devices, the personal exposure data were about 30% of the stationary pollen concentrations with significant correlation (log-transformed data, r = 0.719, p <0.0001). Considering the average time the subjects spent outdoors (14% of sampling time), the indoor-outdoor ratio for pollen was 0.2. In conclusion, pollen reports are reliable for estimating personal exposure over a limited time period although personal pollen exposure is much lower.

  3. Simultaneous allergy to vine pollen and grape.

    PubMed

    Mur, P; Feo Brito, F; Bartolomé, B; Galindo, P A; Gómez, E; Borja, J; Alonso, A

    2006-01-01

    We report the case of an 18-year-old female student suffering from seasonal rhinoconjunctivitis with sensitization to pollens from vine and also from grass, olive, and Chenopodiaceae plants who had recently developed episodes of itching, maculopapular rash, and facial angioedema after eating grapes. Testing revealed positive reactions to vine pollen and grapes, and specific IgE were found for both allergens. Immunoblotting and inhibition assays revealed cross-reactivity between the allergenic structures of vine pollen and grape fruit and also among botanically unrelated pollens.

  4. [Chemical composition of fresh bee pollen collected in the Misintá páramo from the Venezuelan Andes].

    PubMed

    Vit, Patricia; Santiago, B

    2008-12-01

    Venezuelan bee pollen has not been characterized, and marketing is not regulated. Pollen is consumed for apitherapeutical purposes for its nutritional and medicinal properties. This product of the hive is the most popular after honey; therefore it is necessary to characterize and to value it to initiate a database to support the proposal of a norm for bee pollen quality control. Samples of bee pollen collected by bees in the Misintá páramo of Mérida state were characterized accoridng to the chemical composition (moisture, ash, fat, pH, proteins) of four color fractions (yellow, orange, ochre, green). Yellow pollen was the most frequent fraction, with 2.18 g ash/100 g, 5.37 g ether extract/100 g, 14.88 g moisture/100 g, and 37.32 g proteins/100 g.

  5. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes

    PubMed Central

    Verde, Ignacio; Vandecasteele, Grégoire; Lezoualc'h, Frank; Fischmeister, Rodolphe

    1999-01-01

    The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (ICa) and intracellular cyclic AMP concentration ([cAMP]i) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT–PCR.IBMX (100 μM), a broad-spectrum PDE inhibitor, increased basal ICa by 120% and [cAMP]i by 70%, similarly to a saturating concentration of the β-adrenoceptor agonist isoprenaline (1 μM). However, MIMX (1 μM), a PDE1 inhibitor, EHNA (10 μM), a PDE2 inhibitor, cilostamide (0.1 μM), a PDE3 inhibitor, or Ro 20-1724 (0.1 μM), a PDE4 inhibitor, had no effect on basal ICa and little stimulatory effects on [cAMP]i (20–30%).Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on ICa or [cAMP]i. While all combinations tested significantly increased [cAMP]i (40–50%), only cilostamide (0.1 μM)+Ro20-1724 (0.1 μM) produced a significant stimulation of ICa (50%). Addition of EHNA (10 μM) to this mix increased ICa to 110% and [cAMP]i to 70% above basal, i.e. to similar levels as obtained with IBMX (100 μM) or isoprenaline (1 μM).When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased ICa by (≈40% and had negligible effect on [cAMP]i, each selective PDE inhibitor induced a clear stimulation of [cAMP]i and an additional increase in ICa. Maximal effects on ICa were ≈8% for MIMX (3 μM), ≈20% for EHNA (1–3 μM), ≈30% for cilostamide (0.3–1 μM) and ≈50% for Ro20-1724 (0.1 μM).Our results demonstrate that PDE1-4 subtypes regulate ICa in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal ICa, all four PDE subtypes determine the response of ICa to a stimulus activating cyclic AMP production, with the rank order of potency PDE4>PDE3

  6. Nucleotide Metabolism and DNA Replication.

    PubMed

    Warner, Digby F; Evans, Joanna C; Mizrahi, Valerie

    2014-10-01

    The development and application of a highly versatile suite of tools for mycobacterial genetics, coupled with widespread use of "omics" approaches to elucidate the structure, function, and regulation of mycobacterial proteins, has led to spectacular advances in our understanding of the metabolism and physiology of mycobacteria. In this article, we provide an update on nucleotide metabolism and DNA replication in mycobacteria, highlighting key findings from the past 10 to 15 years. In the first section, we focus on nucleotide metabolism, ranging from the biosynthesis, salvage, and interconversion of purine and pyrimidine ribonucleotides to the formation of deoxyribonucleotides. The second part of the article is devoted to DNA replication, with a focus on replication initiation and elongation, as well as DNA unwinding. We provide an overview of replication fidelity and mutation rates in mycobacteria and summarize evidence suggesting that DNA replication occurs during states of low metabolic activity, and conclude by suggesting directions for future research to address key outstanding questions. Although this article focuses primarily on observations from Mycobacterium tuberculosis, it is interspersed, where appropriate, with insights from, and comparisons with, other mycobacterial species as well as better characterized bacterial models such as Escherichia coli. Finally, a common theme underlying almost all studies of mycobacterial metabolism is the potential to identify and validate functions or pathways that can be exploited for tuberculosis drug discovery. In this context, we have specifically highlighted those processes in mycobacterial DNA replication that might satisfy this critical requirement.

  7. Transcriptional mapping and nucleotide sequence of the Listeria monocytogenes hlyA region reveal structural features that may be involved in regulation.

    PubMed Central

    Mengaud, J; Vicente, M F; Cossart, P

    1989-01-01

    DNA sequence analysis of the regions adjacent to the hlyA gene, which encodes listeriolysin O, an essential virulence factor of Listeria monocytogenes, revealed the presence of two open reading frames (ORFs): ORF D located 304 base pairs downstream from hlyA, and ORF U located 224 base pairs upstream from and in opposite direction to hlyA. Promoter mapping performed with RNAs extracted from cells growing exponentially in rich medium showed that the three ORFs are independently transcribed. hlyA is transcribed from two promoters separated by 10 base pairs (P1 hlyA and P2 hlyA). ORF U is transcribed in the opposite direction from an adjacent promoter. These two promoter regions are separated by a palindromic sequence T-T-A-A-C-A-A/T-T-G-T-T-A-A. This palindrome was also found upstream from the ORF D promoter, suggesting that all three genes are similarly regulated. Images PMID:2509367

  8. Anther-preferential expressing gene PMR is essential for the mitosis of pollen development in rice.

    PubMed

    Liu, Yaqin; Xu, Ya; Ling, Sheng; Liu, Shasha; Yao, Jialing

    2017-06-01

    Phenotype identification, expression examination, and function prediction declared that the anther-preferential expressing gene PMR may participate in regulation of male gametophyte development in rice. Male germline development in flowering plants produces the pair of sperm cells for double fertilization and the pollen mitosis is a key process of it. Although the structural features of male gametophyte have been defined, the molecular mechanisms regulating the mitotic cell cycle are not well elucidated in rice. Here, we reported an anther-preferential expressing gene in rice, PMR (Pollen Mitosis Relative), playing an essential role in male gametogenesis. When PMR gene was suppressed via RNAi, the mitosis of microspore was severely damaged, and the plants formed unmatured pollens containing only one or two nucleuses at the anthesis, ultimately leading to serious reduction of pollen fertility and seed-setting. The CRISPR mutants, pmr-1 and pmr-2, both showed the similar defects as the PMR-RNAi lines. Further analysis revealed that PMR together with its co-expressing genes were liable to participate in the regulation of DNA metabolism in the nucleus, and affected the activities of some enzymes related to the cell cycle. We finally discussed that unknown protein PMR contained the PHD, SWIB and Plus-3 domains and they might have coordinating functions in regulation pathway of the pollen mitosis in rice.

  9. The Plant Ovule Secretome: A Different View toward Pollen-Pistil Interactions.

    PubMed

    Liu, Yang; Joly, Valentin; Dorion, Sonia; Rivoal, Jean; Matton, Daniel P

    2015-11-06

    During plant sexual reproduction, continuous exchange of signals between the pollen and the pistil (stigma, style, and ovary) plays important roles in pollen recognition and selection, establishing breeding barriers and, ultimately, leading to optimal seed set. After navigating through the stigma and the style, pollen tubes (PTs) reach their final destination, the ovule. This ultimate step is also regulated by numerous signals emanating from the embryo sac (ES) of the ovule. These signals encompass a wide variety of molecules, but species-specificity of the pollen-ovule interaction relies mainly on secreted proteins and their receptors. Isolation of candidate genes involved in pollen-pistil interactions has mainly relied on transcriptomic approaches, overlooking potential post-transcriptional regulation. To address this issue, ovule exudates were collected from the wild potato species Solanum chacoense using a tissue-free gravity-extraction method (tf-GEM). Combined RNA-seq and mass spectrometry-based proteomics led to the identification of 305 secreted proteins, of which 58% were ovule-specific. Comparative analyses using mature ovules (attracting PTs) and immature ovules (not attracting PTs) revealed that the last maturation step of ES development affected almost half of the ovule secretome. Of 128 upregulated proteins in anthesis stage, 106 were not regulated at the mRNA level, emphasizing the importance of post-transcriptional regulation in reproductive development.

  10. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    NASA Technical Reports Server (NTRS)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  11. A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats

    NASA Technical Reports Server (NTRS)

    Safadi, F.; Reddy, V. S.; Reddy, A. S.

    2000-01-01

    Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

  12. Potential genotype-specific single nucleotide polymorphism interaction of common variation in p53 and its negative regulator mdm2 in cholangiocarcinoma susceptibility.

    PubMed

    Zimmer, Vincent; Höblinger, Aksana; Mihalache, Florentina; Assmann, Gunter; Acalovschi, Monica; Lammert, Frank

    2012-07-01

    Aberrant cell cycle control and apoptosis deregulation are involved in biliary carcinogenesis. The tumor suppressor gene p53 and its key negative regulator murine double minute 2 (mdm2) cooperate in modulating these basic cell functions and germline p53 alteration promotes cholangiocarcinoma (CCA) formation in animal models. The potential association between common functional genetic variation in p53 (SNP72 G/C) and mdm2 (SNP309 T/G) and susceptibility to bile duct cancer, however, has not been studied. p53/SNP72 G/C (rs1042522) and mdm2/SNP309 T/G (rs2279744) were genotyped in 182 Caucasian CCA patients and 350 controls using TaqMan assays. Allelic and genotypic differences, including exploratory data analyses (according to gender, tumor localization, early onset and genotypic interactions) were compared in contingency tables using the χ(2) and Fisher's exact tests. The overall comparison of allele and genotype frequencies yielded no significant association between either SNP and CCA susceptibility. Similarly, gender- and localization-specific analyses did not reveal deviations in allelic or genotypic distributions. In carriers of the low-apoptotic p53 genotype CC, the mdm2 SNP309 T allele conferred borderline significant CCA risk [P=0.049; odds ratio (OR), 4.36; 95% CI, 0.92-20.77]. Power analysis confirmed adequate statistical power to exclude major SNP effects (each >97% for OR 1.7). Collectively, the results we obtained from the largest European CCA cohort do not support the hypothesis of a prominent role of common p53 and mdm2 variation in the genetic susceptibility to bile duct cancer. However, epistatic effects may modulate genetic CCA risk in individual subsets.

  13. Potential genotype-specific single nucleotide polymorphism interaction of common variation in p53 and its negative regulator mdm2 in cholangiocarcinoma susceptibility

    PubMed Central

    ZIMMER, VINCENT; HÖBLINGER, AKSANA; MIHALACHE, FLORENTINA; ASSMANN, GUNTER; ACALOVSCHI, MONICA; LAMMERT, FRANK

    2012-01-01

    Aberrant cell cycle control and apoptosis deregulation are involved in biliary carcinogenesis. The tumor suppressor gene p53 and its key negative regulator murine double minute 2 (mdm2) cooperate in modulating these basic cell functions and germline p53 alteration promotes cholangiocarcinoma (CCA) formation in animal models. The potential association between common functional genetic variation in p53 (SNP72 G/C) and mdm2 (SNP309 T/G) and susceptibility to bile duct cancer, however, has not been studied. p53/SNP72 G/C (rs1042522) and mdm2/SNP309 T/G (rs2279744) were genotyped in 182 Caucasian CCA patients and 350 controls using TaqMan assays. Allelic and genotypic differences, including exploratory data analyses (according to gender, tumor localization, early onset and genotypic interactions) were compared in contingency tables using the χ2 and Fisher’s exact tests. The overall comparison of allele and genotype frequencies yielded no significant association between either SNP and CCA susceptibility. Similarly, gender- and localization-specific analyses did not reveal deviations in allelic or genotypic distributions. In carriers of the low-apoptotic p53 genotype CC, the mdm2 SNP309 T allele conferred borderline significant CCA risk [P=0.049; odds ratio (OR), 4.36; 95% CI, 0.92–20.77]. Power analysis confirmed adequate statistical power to exclude major SNP effects (each >97% for OR 1.7). Collectively, the results we obtained from the largest European CCA cohort do not support the hypothesis of a prominent role of common p53 and mdm2 variation in the genetic susceptibility to bile duct cancer. However, epistatic effects may modulate genetic CCA risk in individual subsets. PMID:22807971

  14. High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen.

    PubMed

    Safavian, Darya; Jamshed, Muhammad; Sankaranarayanan, Subramanian; Indriolo, Emily; Samuel, Marcus A; Goring, Daphne R

    2014-09-01

    We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thaliana Exo70A1's expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1's function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1's proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions.

  15. Nucleotide signalling during inflammation

    PubMed Central

    Idzko, Marco; Ferrari, Davide; Eltzschig, Holger K.

    2014-01-01

    Inflammatory conditions are associated with the extracellular release of nucleotides, particularly ATP. In the extracellular compartment, ATP predominantly functions as a signalling molecule through the activation of purinergic P2 receptors. Metabotropic P2Y receptors are G-protein-coupled, whereas ionotropic P2X receptors are ATP-gated ion channels. Here we discuss how signalling events through P2 receptors alter the outcomes of inflammatory or infectious diseases. Recent studies implicate a role for P2X/P2Ysignalling in mounting appropriate inflammatory responses critical for host defence against invading pathogens or tumours. Conversely, P2X/P2Y signalling can promote chronic inflammation during ischaemia and reperfusion injury, inflammatory bowel disease or acute and chronic diseases of the lungs. Although nucleotide signalling has been used clinically in patients before, research indicates an expanding field of opportunities for specifically targeting individual P2 receptors for the treatment of inflammatory or infectious diseases. PMID:24828189

  16. Aerodynamics and pollen ultrastructure in Ephedra.

    PubMed

    Bolinder, Kristina; Niklas, Karl J; Rydin, Catarina

    2015-03-01

    Pollen dispersal is affected by the terminal settling velocity (Ut) of the grains, which is determined by their size, bulk density, and by atmospheric conditions. The likelihood that wind-dispersed pollen is captured by ovulate organs is influenced by the aerodynamic environment created around and by ovulate organs. We investigated pollen ultrastructure and Ut of Ephedra foeminea (purported to be entomophilous), and simulated the capture efficiency of its ovules. Results were compared with those from previously studied anemophilous Ephedra species.• Ut was determined using stroboscopic photography of pollen in free fall. The acceleration field around an "average" ovule was calculated, and inflight behavior of pollen grains was predicted using computer simulations. Pollen morphology and ultrastructure were investigated using SEM and STEM.• Pollen wall ultrastructure was correlated with Ut in Ephedra. The relative proportion and amount of granules in the infratectum determine pollen bulk densities, and (together with overall size) determine Ut and thus dispersal capability. Computer simulations failed to reveal any functional traits favoring anemophilous pollen capture in E. foeminea.• The fast Ut and dense ultrastructure of E. foeminea pollen are consistent with functional traits that distinguish entomophilous species from anemophilous species. In anemophilous Ephedra species, ovulate organs create an aerodynamic microenvironment that directs airborne pollen to the pollination drops. In E. foeminea, no such microenvironment is created. Ephedroid palynomorphs from the Cretaceous share the ultrastructural characteristics of E. foeminea, and at least some may, therefore, have been produced by insect-pollinated plants. © 2015 Botanical Society of America, Inc.

  17. Pollen foraging behaviour of solitary Hawaiian bees revealed through molecular pollen analysis.

    PubMed

    Wilson, Erin E; Sidhu, C Sheena; LeVan, Katherine E; Holway, David A

    2010-11-01

    Obtaining quantitative information concerning pollinator behaviour has become a primary objective of pollination studies, but methodological limitations hinder progress towards this goal. Here, we use molecular genetic methods in an ecological context to demonstrate that endemic Hawaiian Hylaeus bees (Hymenoptera: Colletidae) selectively collect pollen from native plant species in Haleakala and Hawaii Volcanoes National Parks. We identified pollen DNA from the crops (internal storage organs) of 21 Hylaeus specimens stored in ethanol for up to 3 years. Genetic analyses reveal high fidelity in pollen foraging despite the availability of pollen from multiple plant species present at each study site. At high elevations in Haleakala, pollen was available from more than 12 species of flowering plants, but Hawaiian silversword (Argyroxiphium sandwicense subsp. macrocephalum) comprised 86% of all pollen samples removed from bee crops. At lower elevations in both parks, we only detected pukiawe (Leptecophylla (Styphelia) tameiameiae) pollen in Hylaeus crops despite the presence of other plant species in flower during our study. Furthermore, 100% of Hylaeus crops from which we successfully identified pollen contained native plant pollen. The molecular approaches developed in this study provide species-level information about floral visitation of Hawaiian Hylaeus that does not require specialized palynological expertise needed for high-throughput visual pollen identification. Building upon this approach, future studies can thus develop appropriate and customized criteria for assessing mixed pollen loads from a broader range of sources and from other global regions. © 2010 Blackwell Publishing Ltd.

  18. Specialized bees fail to develop on non-host pollen: do plants chemically protect their pollen?

    PubMed

    Praz, Christophe J; Müller, Andreas; Dorn, Silvia

    2008-03-01

    Bees require large amounts of pollen for their own reproduction. While several morphological flower traits are known to have evolved to protect plants against excessive pollen harvesting by bees, little is known