Hormonal Regulation of Gametangial Formation in the Moss Bryum argenteum Hedw

Phytohormones such as auxins, cytokinins, gibberellins, andabscisic acid differentially affect gametangial induction inmale and female clones of Bryum argenteum. Both IAA and GA³increased the percentage of fertile gametophores in the maleclone, and inhibited the response in the female clone. GA₃ wasmore effective than IAA in eliciting the response in the maleclone. Cytokinins, on the other hand, increased the productionof fertile gametophores in the female clone, and inhibited itslightly in the male clone. The two cytokinins tested (kinetinand DMAAP) were almost equally effective for the female clone. An Interaction of IAA and kinetin nullified their individualinhibitory effects on the female and male clones, respectively.Cyclic AMP prevented the inhibitory effect of kinetin in themale clone; whereas, in the female clone, it stimulated theresponse elicited by kinetin. Abscisic acid (ABA) acted as ageneral inhibitor of vegetative growth and gametangial inductionin this moss. However, the inhibition of gametangial inductionwas greater in the female clone which is also more sensitiveto ABA than the male clone.     

Role of the Mitochondrial Permeability Transition Pore in Apoptosis

Mitochondrial permeability transition (PT) involves the formation of proteaceous, regulated pores, probably by apposition of inner and outer mitochondrial membrane proteins which cooperate to form the mitochondrial megachannel (=mitochondrial PT pore). PT has important metabolic consequences, namely the collapse of the mitochondrial transmembrane potential, uncoupling of the respiratory chain, hyperproduction of superoxide anions, disruption of mitochondrial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins. Recent evidence suggests that PT is a critical, rate limiting event of apoptosis (programmed cell death): (i) induction of PT suffices to cause apoptosis; (ii) one of the immediate consequences of PT, disruption of the mitochondrial transmembrane potential (_m), is a constant feature of early apoptosis; (iii) prevention of PT impedes the _m collapse as well as all other features of apoptosis at the levels of the cytoplasma, the nucleus, and the plasma membrane; (iv) PT is modulated by members of the apoptosis-regulatory bcl-2 gene family. Recent data suggest that the acquisition of the apoptotic phenotype, including characteristic changes in nuclear morphology and biochemistry (chromatin condensation and DNA fragmentation), depends on the action of apoptogenic proteins released from the mitochondrial intermembrane space.     

Variations in Fusion Pore Formation in Cholesterol-Treated Platelets

Exocytosis is a highly regulated intercellular communication process involving various membrane proteins, lipids, and cytoskeleton restructuring. These components help control granule fusion with the cell membrane, creating a pore through which granular contents are released into the extracellular environment. Platelets are an ideal model system for studying exocytosis due to their lack of a nucleus, resulting in decreased membrane regulation in response to cellular changes. In addition, platelets contain fewer granules than most other exocytosing cells, allowing straightforward measurement of individual granule release with carbon-fiber microelectrode amperometry. This technique monitors the concentration of serotonin, an electroactive molecule found in the dense-body granules of platelets, released as a function of time, with 50 μs time resolution, revealing biophysical characteristics of the fundamental exocytotic process. Variations in fusion pore formation and closure cause deviations from the classic current versus time spike profile and may influence diffusion of serotonin molecules from the site of granule fusion. Physiologically, the delivery of smaller packets of chemical messengers or the prolonged delivery of chemical messengers may represent how cells/organisms tune biological response. The goals of this work are twofold: 1) to categorize secretion features that deviate from the traditional mode of secretion and 2) to examine how changing the cholesterol composition of the platelet membrane results in changes in the pore formation process. Results herein indicate that the expected traditional mode of release is actually in the minority of granule content release events. In addition, results indicate that as the cholesterol content of the plasma membrane is increased, pore opening is less continuous.     

The Energetics of Membrane Fusion from Binding,through Hemifusion,Pore Formation,and Pore Enlargement

The main steps of viral membrane fusion are local membrane approach, hemifusion, pore formation, and pore enlargement. Experiments and theoretical analyses have helped determine the relative energies required for each step. Key protein structures and conformational changes of the fusion process have been identified. The physical deformations of monolayer bending and lipid tilt have been applied to the steps of membrane fusion. Experiment and theory converge to strongly indicate that, contrary to former conceptions, the fusion process is progressively more energetically difficult: hemifusion has a relatively low energy barrier, pore formation is more energy-consuming, and pore enlargement is the most difficult to achieve.This revised version was published online in August 2005 with a corrected cover date.     

Sporophyte Formation and Life Cycle Completion in Moss Requires Heterotrimeric G-Proteins

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.In all known eukaryotes, cellular signaling involves heterotrimeric GTP-binding proteins (G-proteins), which consist of Gα, Gβ, and Gγ subunits (Cabrera-Vera et al., 2003). According to the established paradigm, when Gα is GDP-bound, it forms a trimeric complex with the Gβγ dimer and remains associated with a G-protein coupled receptor. Signal perception by the receptor facilitates GDP to GTP exchange on Gα. GTP-Gα dissociates from the Gβγ dimer, and both these entities can transduce the signal by interacting with different effectors. The duration of the active state is determined by the intrinsic GTPase activity of Gα, which hydrolyzes bound GTP into GDP and inorganic phosphate (Pi), followed by the reassociation of the inactive, trimeric complex (Siderovski and Willard, 2005).In plants, G-protein signaling has been studied in only a few angiosperms to date at the functional level, although the proteins exist in the entire plant lineage (Hackenberg and Pandey, 2014; Urano and Jones, 2014; Hackenberg et al., 2016). Interestingly, while the overall biochemistry of the individual G-protein components and the interactions between them are conserved between plant and metazoan systems, deviations from the established norm are also obvious. For example, the repertoire of canonical G-proteins is significantly limited in plants; the human genome codes for 23 Gα, 5 Gβ, and 12 Gγ proteins, whereas most plant genomes, including those of basal plants, typically encode 1 canonical Gα, 1 Gβ, and three to five Gγ proteins (Urano and Jones, 2014). The only exceptions are some polyploid species, such as soybean, which have retained most of the duplicated G-protein genes (Bisht et al., 2011; Choudhury et al., 2011). Moreover, even in plants that possess only a single canonical Gα and Gβ protein, for example Arabidopsis (Arabidopsis thaliana) and rice, the phenotypes of plants lacking either one or both proteins are relatively subtle. The mutant plants exhibit multiple developmental and signaling defects but are able to complete the life cycle without any major consequences. These observations have questioned the significance of G-protein mediated signaling pathways in plants.Interestingly, plants also possess certain unique variants of the classical G-protein components such as the type III Cys-rich Gγ proteins and extra-large GTP-binding (XLG) proteins, which add to the diversity and expanse of the G-protein signaling networks (Roy Choudhury et al., 2011; Chakravorty et al., 2015; Maruta et al., 2015). The XLG proteins are almost twice the size of typical Gα proteins, with the C-terminal region that codes for Gα-like domain and an extended N-terminal region without any distinctive features. Plant XLGs are encoded by entirely independent genes and therefore are different from the mammalian extra-long versions of Gα proteins such as XLαs and XXLαs, which are expressed due to the use of alternate exons (Abramowitz et al., 2004). Three to five copies of XLG proteins are present in the genome of most angiosperms. At the functional level, the XLG proteins have been characterized only from Arabidopsis, to date, where recent studies suggest that the proteins compete with canonical Gα for binding with the Gβγ dimers and may form functional trimeric complexes (Chakravorty et al., 2015; Maruta et al., 2015). The XLG and Gβγ mutants of Arabidopsis seem to function in the same pathways during the regulation of a subset of plant responses, for example primary root length and its regulation by abscisic acid (ABA); the root waving and skewing responses; sensitivity to Glc, salt, and tunicamycin; and sensitivity to certain bacterial and fungal pathogens (Ding et al., 2008; Pandey et al., 2008; Chakravorty et al., 2015; Maruta et al., 2015). However, many of the phenotypes of Arabidopsis Gα and Gβγ mutants are also distinct from that of the xlg triple mutants. For example, compared to the wild-type plants, the canonical G-protein mutants exhibit altered response to gibberellic acid, brassinosteroids, and auxin and show changes in leaf shape, branching, flowering time, and stomatal densities (Ullah et al., 2003; Chen et al., 2004; Pandey et al., 2006; Zhang et al., 2008; Nilson and Assmann, 2010). The xlg triple mutants behave similarly to wild-type plants in all these aspects of development and signaling. Moreover, whether the XLG proteins are authentic GTP-binding and -hydrolyzing proteins and the extent to which they directly participate in G-protein-mediated signaling pathways remains confounding (Chakravorty et al., 2015; Maruta et al., 2015). Even in plants with a limited number of G-protein subunits such as Arabidopsis, one Gα and three XLGs potentially compete for a single Gβ protein, and the analysis of null mutants is not straightforward, that is, it is not possible to delineate whether the phenotypes seen in the Gα null mutants are truly due to the lack of Gα and/or because of an altered stoichiometry or availability of Gβ for the XLG proteins.As a bryophyte, Physcomitrella patens occupies a unique position in the evolutionary history of plants. It lacks vasculature but exhibits alteration between generations, which is dominated by a gametophytic (haploid) phase and a short sporophytic (diploid) phase (Cove et al., 2009). Many of the pathways related to hormone signaling, stress responses, and development are conserved between angiosperms and P. patens (Cove et al., 2009; Sun, 2011; Komatsu et al., 2013; Yasumura et al., 2015). It is also an intriguing example in the context of the G-protein signaling, because its fully sequenced genome does not encode a canonical Gα gene, although genes coding for the Gβ and Gγ proteins exist. A single gene for a potential XLG homolog also exists in the P. patens genome. This unique assortment of proteins predicts several alternative scenarios for G-protein signaling in P. patens. For example, the P. patens Gβγ proteins might be nonfunctional due to the loss of canonical Gα and are left in the genome as evolutionary artifacts. Alternatively, the Gβγ proteins of P. patens might maintain functionality regardless of the existence of a canonical Gα protein in pathways not regulated via classic G-protein signaling modes. Finally, a more likely scenario could be that the potential XLG protein can substitute for the Gα function in P. patens.To explore these possibilities and understand better the conserved and unique mechanisms of G-protein signaling pathways in plants and their significance, we examined the role of G-protein subunits in P. patens. We provide unambiguous evidence for the genetic coupling of XLG and Gβ proteins in controlling P. patens development. In contrast to all other plant species analyzed to date, where G-proteins are not essential for growth and survival, the XLG or one of the Gβ proteins is required for the sporophyte formation and life cycle completion in P. patens. Furthermore, one of the Arabidopsis XLG proteins, XLG2, and the canonical Gβ protein AGB1 can functionally complement the P. patens mutant phenotypes. These data provide new insights in the evolutionary breadth and the spectrum of signaling pathways regulated by G-proteins in plants.     

The Use of Cellulase in Inhibiting Biofilm Formation from Organisms Commonly Found on Medical Implants

A study was made of the use of cellulase to inhibit biofilm formation by a pathogenic bacterium commonly found in medical implants. A Pseudomonas aeruginosa biofilm was grown on glass slides in a parallel flow chamber for 4 d with glucose as the nutrient source. Biofilm development was assessed by measuring the colony forming units (CFU) and biomass areal density. Biofilm was grown at pH 5 and 7 in the presence of three different cellulase concentrations, 9.4, 37.6 and 75.2 units ml^{m 1}. In addition, a control study using deactivated cellulase was performed. The results show that cellulase is effective in partially inhibiting biomass and CFU formation by P. aeruginosa on glass surfaces. The effect of cellulase depended on concentration and was more effective at pH 5 than pH 7. The experiment was further extended by investigating the effect of cellulase on the apparent molecular weight of purified P. aeruginosa exopolysaccharides (EPS). The observation of EPS using size exclusion chromatography showed a decrease in apparent molecular weight when incubated with enzyme. An increase in the amount of reducing sugar with time when the purified EPS were incubated with enzyme also supports the hypothesis that cellulase degrades the EPS of P. aeruginosa. While cellulase does not provide total inhibition of biofilm formation, it is possible that the enzyme could be used in combination with other treatments or in combinations with other enzymes to increase effectiveness.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

Imaging the Lipid-Phase-Dependent Pore Formation of Equinatoxin II in Droplet Interface Bilayers

Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.     

Atomistic Simulations of Pore Formation and Closure in Lipid Bilayers

Cellular membranes separate distinct aqueous compartments, but can be breached by transient hydrophilic pores. A large energetic cost prevents pore formation, which is largely dependent on the composition and structure of the lipid bilayer. The softness of bilayers and the disordered structure of pores make their characterization difficult. We use molecular-dynamics simulations with atomistic detail to study the thermodynamics, kinetics, and mechanism of pore formation and closure in DLPC, DMPC, and DPPC bilayers, with pore formation free energies of 17, 45, and 78 kJ/mol, respectively. By using atomistic computer simulations, we are able to determine not only the free energy for pore formation, but also the enthalpy and entropy, which yields what is believed to be significant new insights in the molecular driving forces behind membrane defects. The free energy cost for pore formation is due to a large unfavorable entropic contribution and a favorable change in enthalpy. Changes in hydrogen bonding patterns occur, with increased lipid-water interactions, and fewer water-water hydrogen bonds, but the total number of overall hydrogen bonds is constant. Equilibrium pore formation is directly observed in the thin DLPC lipid bilayer. Multiple long timescale simulations of pore closure are used to predict pore lifetimes. Our results are important for biological applications, including the activity of antimicrobial peptides and a better understanding of membrane protein folding, and improve our understanding of the fundamental physicochemical nature of membranes.     

福建药用苔鲜植物资源

本文报道了福建药用苔鲜植物20种,并对它们的生境、怪味、功效等作了初步分析。     

基因定点整合技术及其在苔藓研究中的进展

基因定点整合技术是20世纪80年代后兴起的一种分子生物学技术,在精细研究基因功能,消除转基因沉默,基因治疗等有重要意义。基因定点整合技术是功能基因组学研究的重要手段。目前关于基因定点整合技术在酵母和鼠胚胎干细胞中的应用已经很成熟,高等植物由于同源重组频率较低而限制了它的应用,但小立碗藓(Physcomitrella patens)基因同源重组频率较高,基因定点整合技术得到了成功应用,可望成为一种新的分子生物学的模式植物。本文针对基因定点整合的原理、技术路线及进展作一综述。     

Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.     

The Role of Meristematic Activities in the Formation of Leaf Blades

Arabidopsis thaliana (L.) Heynh. Leaf primordia also have their own meristematic regions and meristematic activity is maintained in part of the leaf blade, in some case, even after maturation. Transgenic plants have been generated that have proved to be useful tools in the analysis of the behavior of meristematic regions in leaf blades of A. thaliana. This review, based on our present understanding of molecular mechanisms for the maintenance and development of shoot apical meristems in A. thaliana, summarizes the variations in patterns and functions of meristematic regions in leaf blades focusing, in particular, on the case of indeterminate leaves. Received 5 April 2000/ Accepted in revised form 12 April 2000     

The Role of Cell Hydrophobicity in the Formation of Aerobic Granules

Cell hydrophobicity is an important affinity force in cell self-immobilization and attachment processes. The role of cell hydrophobicity in the formation of aerobic granules has not been clear. Therefore, two series of experiments were conducted to investigate the role of cell hydrophobicity in the formation of aerobic heterotrophic and nitrifying granules in sequencing batch reactors, while the effects of shear strength, hydraulic selection pressure, and organic loading rate on the cell hydrophobicity were also studied. Results showed that the formations of heterotrophic and nitrifying granules were associated very closely with the cell hydrophobicity. The hydrophobicity of granular sludge was nearly twofold higher than that of conventional bioflocs. A high shear force or hydraulic selection pressure imposed on microorganisms resulted in a significant increase in the cell hydrophobicity, while the cell hydrophobicity seemed not to be sensitive to the changes in the organic loading rates in the range studied. In conclusion, the cell hydrophobicity could induce and further strengthen cell–cell interaction, and might be a main triggering force to initiate the granulation of heterotrophic and nitrifying bacteria. Received: 21 May 2002 / Accepted: 21 June 2002     

The Role of Structural Water in the Formation of Nucleotide Mispairs

Abstract

The results of NMR investigation of the double-helical nucleic acid fragments containing A · C, C · U, m⁶G · U and m⁶G · G mispairs can be explained on the assumption that the bases in such pairs being in usual tautomeric forms are linked via water bridges. A computer analysis of intermolecular interactions in the systems containing two bases and one or two water molecules shows that these pairs correspond to the energy minima. The formation of pairs with water bridges can be considered an intermediate step in mutagenesis caused by some spontaneous errors arising during nucleic acid biosynthesis as well in mutagenesis induced by alkylating agents.     

The Role of Cellulase and Polygalacturonase in Abscission of Young and Mature Shamouti Orange Fruits

During the first eight weeks after setting young citrus fruits gradually lose their ability to abscise at the abscission zone between the stem and the pedicel; in fruits older than eight weeks abscission occurs at the calyx area. The activity of cellulase and polygalacturonase in the two abscission zones was markedly increased before and during abscission, and was localized mainly in the abscission zone. Ethylene accelerated the increase in enzymic activity after an 8- to 10-h lag period; 2,4-D delayed abscission and enzymic activity when applied during the first 24 h after excision. During this period 2,4-D also partly suppressed the enhancing effect of ethylene. Early application of cyclo-heximide inhibited the formation of the enzymes and thus abscission was delayed to a certain extent. Although there are some indications that the relationship between enzymic activity and abscission is a complex one, the data presented indicate that cellulase and polygalacturonase play a significant role in abscission of citrus fruits at various developmental stages. Both enzymes act almost simultaneously and are equally controlled by ethylene and 2,4-D.     

Effect of Cultural Conditions on Cellulase Formation by Trichoderma viride

In order to have increased extracellular production of cellulase by Trichoderma viride ITCC 1433, the organism was grown on various growth factors. Cellulose Powder ?123 was found to be the best C-source while amongst raw materials, alkali-treated rice straw gave the best yield. A combination of peptone, urea and ammonium sulphate gave better production of cellulase than when a single nitrogen source was used. Sugars when added into the cellulose medium, generally suppressed the yield. When the organism was grown on sugars as the sole source of carbon, only lactose and maltose induced any cellulase production. Acetate and ascorbate were conspicuous in increasing cellulase production and when given together they had a cummulative effect and the yeild was doubled.     

Effect of Chelating Agents and Metal Ions on Gametangial Formation in the Moss Bryum argenteum Hedw

Chelating agents such as EDTA and EDDHA markedly stimulate theformation of gametangia in the dioecious moss Bryum argenteum,and the effect is more pronounced on male than on female clones.EDTA-stimulated gametangial formation is associated with significantincreases in endogenous iron and copper. Ferric citrate alsoenhances gametangial formation, but copper sulphate is inhibitory.The present findings are discussed in the context of earlierinvestigations on other plants in an attempt to explain thepossible involvement of chelating agents and metal ions in stimulatingthe onset of the reproductive phase in this moss. Bryum argenteum, gametangial formation, chelating agents, metal ions