Proteomic analysis of the mode of antibacterial action of silver nanoparticles

Silver nanoparticles (nano-Ag) are potent and broad-spectrum antimicrobial agents. In this study, spherical nano-Ag (average diameter = 9.3 nm) particles were synthesized using a borohydride reduction method and the mode of their antibacterial action against E. coli was investigated by proteomic approaches (2-DE and MS identification), conducted in parallel to analyses involving solutions of Ag(+) ions. The proteomic data revealed that a short exposure of E. coli cells to antibacterial concentrations of nano-Ag resulted in an accumulation of envelope protein precursors, indicative of the dissipation of proton motive force. Consistent with these proteomic findings, nano-Ag were shown to destabilize the outer membrane, collapse the plasma membrane potential and deplete the levels of intracellular ATP. The mode of action of nano-Ag was also found to be similar to that of Ag(+) ions (e.g., Dibrov, P. et al, Antimicrob. Agents Chemother. 2002, 46, 2668-2670); however, the effective concentrations of nano-Ag and Ag(+) ions were at nanomolar and micromolar levels, respectively. Nano-Ag appear to be an efficient physicochemical system conferring antimicrobial silver activities.     

Histochemical changes in the developing abscission layer in fruits of Prunus cerasus L.

Summary Abscission layer formation in the sour cherry (Prunus cerasus L.) during fruit maturation occurred in the transition zone between the fruit and the pedicel. The abscission layer, consisting of 5–8 rows of cells, was first identified by its low affinity for haematoxylin. The walls of cells in the abscission layer contained less total polysaccharides than adjacent cells. The pectins were degraded and the cellulose was partially broken down resulting in cell separation. The Ca level in the abscission zone decreased and Ca and Mg were lost from the walls of cells in the layer during abscission. After the abscission layer formed, cells associated with the layer had a lower capacity to bind ⁴⁵Ca than cells distal or proximal to the layer.Michigan Agricultural Experiment Station Journal Article No. 4607     

Occurrence and Localization of 9.5 Cellulase in Abscising and Nonabscising Tissues

Nitrocellulose tissue prints immunoblotted with 9.5 cellulase antibody were used to demonstrate areas of cellulase localization within Phaseolus vulgaris explants on exposure to ethylene. The 9.5 cellulase was induced in the distal and proximal abscission zone and in the stem. In both abscission zones, the 9.5 cellulase was found in the cortical cells of the separation layer, which develops as a narrow band of cells at the place where fracture occurs. The enzyme was also found associated with the vascular traces of the tissues adjacent to the separation layer extending through the first few millimeters at each side of the separation layer. The two abscission zones differed in the way that cellulase distributed through the separation layer as abscission proceeded. In the distal zone, cellulase appeared first in the cells of the separation layer adjacent to vascular traces and extended toward the periphery. In the proximal zone, 9.5 cellulase accumulated first in the cortical cells that lie in the adaxial side and then extended to the abaxial side. In response to ethylene, 9.5 cellulase was also induced in the vascular traces of the stem and the pulvinus without developing a separation layer. The role of 9.5 cellulase in the vascular traces is unknown. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting with 9.5 cellulase antibody identified the same 51-kilodalton protein in both abscising and nonabscising tissues. Therefore, the determinant characteristic of the abscission process is the induction of 9.5 cellulase by cortical cells in the separation layer, and this implies that these cells have a unique mechanism for initiating 9.5 cellulase synthesis.     

Seasonality of epiphytic development of the hydroid Obelia geniculata on cultivated Saccharina japonica (Laminariaceae, Phaeophyta) in Korea

The occurrence of encrusting colonies of the hydroid Obelia geniculata on farmed Saccharina japonica was examined between December 2007 and July 2008 at a Saccharina farm at Wando on the southwestern coast of Korea. The growth stages of S. japonica can be divided into two phases: an active growth phase from February to the end of May and a decay phase from June to July. There was a significant increase in the level of incrustation by colonies on fronds (measured as the percentage of fronds with encrusting colonies) between February and July (p?<?0.05). The encrusting colonies occurred first on the upper part of the frond in February and progressed to the basal part in July. The abundance of encrusting colonies in relation to the growth phase on farms over time was limited by the harvest of the seaweed crop at the end of the cultivation period in July. The stipes and holdfasts of fronds showed no signs of infestation at any time during the cultivation period. The extent of the infestation appeared to be related to a combination of factors. These could be reduced physiological activity and subsequent tissue aging that occurred simultaneously in the sporophytic life phase of Saccharina frond, and a rapid increase in reproduction and growth of O. geniculata coinciding with rising seawater temperature.     

Alginate content and composition ofMacrocystis pyrifera from New Zealand

Tissue samples ofMacrocystis pyrifera from 2 sites in southern New Zealand and harvested over a period of 12 months were analysed for alginate content and composition (M:G ratio). Plants were divided into three frond classes of different length and each frond was further separated into age categories of blades and stipes (viz young, mature and old blades; mature and old stipes). Within each size class, younger blades had higher alginate content than older blades. Stipes did not show such variation with age. Alginate from younger blades and stipes had higher proportions of mannuronic acid residues than those from old blades and stipes. The range of M:G ratios for age categories of either blades or stipes from longer fronds was greater than those for smaller fronds. Alginate content and M:G ratios of stipes were always higher than for blades. The difference between M:G ratios of blades and stipes was greater for smaller fronds than for longer ones. Differences between collection sites and seasonal trends are also discussed.author for correspondence     

Die induktion eines trenngewebes bei früchten von Prunus avium L. durch 2-Chloräthylphosphonsäure

Summary 2-Chloroethylphosphonic acid (CEPA) facilitates the separation of the fruit from the pedicel significantly. The application of 2,000 and 4,000 ppm CEPA in four sweet cherry varieties during maturation resulted in the formation of a complete abscission layer in the transition zone between pedicel and fruit. In contrast, in the untreated fruit no abscission layer was evident at maturity. The walls of the cells in the abscission layer contained less total polysaccharides than adjacent cells. Cellulose was partially broken down, and the pectins were degraded. The Ca and Mg content in the cell walls decreased. Thus the same histochemical changes are involved in natural and CEPA induced abscission.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Exposure of RBL-2H3 mast cells to Ag(+) induces cell degranulation and mediator release

There is a growing need to understand the impact of environmental sulfhydryl group-reactive heavy metals on the immune system. Here we show that Ag(+) induces mast cell degranulation, as does the aggregation of the high affinity immunoglobulin E receptor (FcepsilonRI). Micromolar quantities of Ag(+) specifically induced degranulation of mast cell model rat basophilic leukemia (RBL-2H3) cells without showing cytotoxicity. The Ag(+)-mediated degranulation could be observed as rapidly as 5 min after the addition of the ions. Ag(+) also induced a rapid change in tyrosine phosphorylation of multiple cellular proteins including the focal adhesion kinase but not Syk kinase. The Syk-selective inhibitor piceatannol and the Src family-selective tyrosine kinase inhibitor PP1 dose-dependently inhibited FcepsilonRI-mediated degranulation, whereas neither compound inhibited the Ag(+)-mediated degranulation. Furthermore, likewise FcepsilonRI aggregation, Ag(+) also induced leukotriene secretion. These results show that Ag(+) activates RBL-2H3 mast cells through a tyrosine phosphorylation-linked mechanism, which is distinct from that involved in FcepsilonRI-mediated activation.     

基于人工培养的浮萍快速无性繁殖营养条件研究

为探究并优化浮萍人工培养技术, 研究以广布种紫萍(Spirodela polyrrhiza)和青萍(Lemna minor)为主要研究对象, 探索两种浮萍植物在不同营养水平的Hoagland和Hunter培养液中的鲜重、叶状体数的生长变化状况。结果表明: (1)紫萍和青萍鲜重在Hoagland培养液的不同营养水平下先增加后减少, 而在Hunter营养液中呈持续增长趋势; 两种浮萍的鲜重最大相对增长率(RGR)分别为0.11和0.18, 鲜重受浮萍品种和培养基类型及其不同营养水平影响显著(P<0.05), 以青萍在Hunter原液培养基下的无性繁殖产生的生物量最高。(2)紫萍和青萍叶状体数在Hoagland培养液的不同营养水平下先增加后减少, 而同鲜重一样在Hunter营养液中呈不断增长趋势; 两种浮萍的叶状体最大相对增长率分别为0.14和0.19, 叶状体生长的RGR变化同样受浮萍品种和培养基类型及营养水平影响显著(P<0.05), 以青萍在Hunter原液的营养环境下收获的叶状体数最高。(3)两种浮萍在Hoagland和Hunter营养液的不同营养水平下鲜重/叶状体比呈下降趋势, 表明两种浮萍在适应不同营养时优先繁殖子代叶状体, 以扩大种的适合度。研究认为Hunter原液可作为广布种青萍的最优培养条件, 可实现短时间内收获较大浮萍鲜生物量和叶状体数, 为进一步资源化利用提供原材料。     

Jasmonates promote abscission in bean petiole expiants: Its relationship to the metabolism of cell wall polysaccharides and cellulase activity

Jasmonic acid (JA) and its methyl ester (JA-Me) promoted the abscission of bean petiole expiants in the dark and light, and the activity of these compounds was almost same. JA and JA-Me did not enhance ethylene production in bean petiole expiants in the light, indicating that the abscission-promoting effects of these compounds are not the result of ethylene. Cells in the petiole adjacent to the abscission zone expanded during abscission but not in the pulvinus, and JA-Me promoted cell expansion in the petiole and the pulvinus. JA-Me had no effect on the total amounts of pectic and hemicellulosic polysaccharides in 2-mm segments of the abscission region, which included 1 mm of pulvinus and 1 mm of petiole from the abscission zone. On the other hand, the total amounts of cellulosic polysaccharides in this region were reduced significantly by the addition of JA-Me in the light. JA-Me had no effect on the neutral sugar composition of hemicellulosic polysaccharides during abscission. The decrease in the endogenous levels of UDP-sugars in the petiole adjacent to the abscission zone was accelerated during abscission by the addition of JA-Me in the light. Cellulase activities of pulvinus and petiole in 10-day-old seedlings were enhanced by the addition of JA. These results suggest that the promoting effect of JA or JA-Me on the abscission of bean petiole explants is due to the change of sugar metabolism in the abscission zone, in which the increase in cellulase activity involves the degradation of cell wall polysaccharides. Jasmonic acid (JA) and its methyl ester (JA-Me) are considered to be putative plant hormones for a number of reasons, including their wide occurrence in the plant kingdom, biologic, activities in multiple aspects at low concentrations, and their interaction with other plant hormones (for reviews see Parthier 1991, Hamberg and Gardner 1992, Sembdner and Parthier 1993, Ueda et al. 1994a). We have already reported that JA and JA-Me and C₁₈-unsaturated fatty acids, which are considered to be the substrates of the biosynthesis of jasmonates, are powerful senescence-promoting substances (Ueda et al. 1982b, 1991a). Senescence symptoms induced by these compounds are identical to those of natural senescence. Recently we have also found that JA inhibited indole-3-acetic acid (IAA)-induced elongation of oat (Avena sativa L. cv. Victory) coleoptile segments by inhibiting the synthesis of cell wall polysaccharides (Ueda et al. 1994b, 1995). These facts led us to study the mode of actions of JA and JA-Me on promoting abscission, which is considered the last dramatic phenomenon of senescence. In this paper we report that JA and JA-Me promote abscission in bean (Phaseolus vulgaris L. cv. Masterpiece) petiole expiants and that the changes in the metabolism of cell wall polysaccharides in the petiole and the pulvinus adjacent to the abscission zone are involved in the promotive effects of these compounds.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - DCB 2,6-dichlorobenzonitrile - HPLC high performance liquid chromatography - IAA indole-3-acetic acid - JA jasmonic acid - JA-Me methyl jasmonate - MES 2-(N-morpholino)ethane-sulfonic acid, monohydrate - TCA trichloroacetic acid - Tris 2-amino-2-hydroxymethy-1,3-propanediole     

Anatomy of leaf abscission in the Amur honeysuckle (Lonicera maackii, Caprifoliaceae): a scanning electron microscopy study

Lonicera maackii (Rupr.) Maxim. (Amur honeysuckle) is native to Asia and an important ornamental in China. However, the anatomy of leaf abscission (shedding) in L. maackii had not been studied previously. Such work is needed not only because knowledge of the leaf abscission process is important for a horticultural species like L. maackii but also because leaf abscission is probably the least understood abscission process, as it occurs so rapidly. Therefore, our objective was to use scanning electron microscopy (SEM) to examine the progression of leaf abscission in L. maackii at the cellular level. L. maackii branches with leaves were regularly collected in Beijing, China over the 2-month period in which leaves abscise, and examined with SEM. We found that, unlike in model species, the cortex is involved in abscission, forming an “abaxial gap.” We discovered that there is no discrete abscission zone prior to the onset of abscission and that no cell divisions precede abscission. An abscission zone did become evident well after the abscission process had begun, but its cells were enlarged, not constricted as in typical abscission zones. In the abaxial gap, intact cells separated at their middle lamella, but in the abscission zone, cell separation involved the entire wall, which is not typical. We did observe expected mechanical fission of vascular tissues. While the leaf abscission process we observed in L. maackii has similarities with model systems, aspects deviate from the expected.     

Spontaneous phloem exudation accompanying abscission in Lupinus mutabilis (Sweet)

Like some of the Mediterranean members of the genus Lupinus, the New World lupin, Lupinus mutabilis (Sweet; cv. Inti), exhibits prolonged (20-40 min) exudation of phloem sap from incisions made in stems, in the raceme and at the tips and sutures of developing fruits. Just prior to or immediately following abscission of flowers of L. mutabilis there was also spontaneous exudation from the proximal face of the abscission zone at the base of the pedicel. This is not a recorded feature of other lupins. Analysis of solutes in this exudate was consistent with its having been derived directly from phloem. The major solutes were sucrose (0.940.04 M), amino acids (18811 mM, 45% as asparagine and 15% as glutamine), K ion (52 mM), and total phosphorus (17 mM). Microscopic examination of the proximal face of the pedicel abscission zone at or following abscission showed little or no breakage of the cells at the zone. The major solutes of spontaneous exudate were similar to those in exudates collected from incisions made in the supporting raceme, upper stem and branches, at the tips and sutures of developing fruits and in the mid- and basal stem regions. However, there were significant compositional differences among minor constituents. The spontaneous exudate had a higher level of Ca ion and, consequently, a narrower Mg/Ca ratio (2.8) than exudate from incisions in the adjacent raceme (9.3) or fruits (15.7). There were also higher concentrations of trace elements (Mn, Zn and Cu) but lower concentrations (3 ng m^-1) of cytokinins compared to exudates collected from incisions (20 ng ml^-1). The relative contents of K and Na ions in exudates from incisions at different sites on the plant showed evidence of selective phloem loading and downward translocation of Na ion and selective loading and upward translocation of K ion.     

The distribution of antigen in lymphoid tissues following its injection into the anterior chamber of the rat eye

Injection of Ag into the anterior chamber (AC) of the eye induces deviant immune responses. It has been proposed that Ag internalized by ocular APCs is presented in a tolerogenic fashion in the spleen. However, the nature and distribution of the Ag-bearing cells in the lymphoid organs remain unclear. Fluorescent-labeled Ag (dextran, BSA) injected into the AC of Lewis rats was detected in the subcapsular sinus of the right submandibular lymph nodes (LNs) and cervical LNs, the marginal zone of the spleen, and the medulla of the mesenteric LNs. In the spleen, Ag-bearing cells were CD1(+), CD11b(+), ED1(+), ED2(low), ED3(+), CD86(low), OX6(+), CD11c(-), ED5(-) and in the LNs were CD4(+), CD8(+), CD80(+), and OX41(+) suggesting these were lymphoid organ resident macrophages. These Ag-bearing macrophages were located adjacent to CD4(+) cells, CD8(+) cells, and NK cells in the LNs and spleen and to marginal zone B cells in the spleen. No interaction with gamma delta T cells was observed. The data demonstrates that Ag derived from the AC of the eye is mainly internalized by resident macrophages in the LNs and spleen which are ideally placed to interact with cells involved in the induction of deviant ocular immune responses. The extensive distribution of Ag in LNs draining the upper airway and gastrointestinal tracts, together with the phenotype of Ag-bearing cells in the lymphoid organs, suggests that Ag leaves the eye predominantly in a soluble form and implies other mechanisms of tolerance may contribute to ocular-specific immune responses.     

A HISTOCHEMICAL STUDY OF ABSCISSION LAYER FORMATION IN THE BEAN

In debladed bean petioles calcium and dry weight increased in the abscission zone during an induction period of 14 hr. Before the microscopic appearance of the abscission layer calcium decreased in the abscission zone and increased in the petiole. Dry matter began to decrease in both the abscission zone and the petiole 24 hr after deblading. The first visual change in the cells of the abscission zone was a swelling of the pectic materials of the cell walls. This was followed by breakdown of other cell wall components, i.e., non-cellulosic polysaccharides and cellulose. The cellulose of the cell walls adjacent and distal to the abscission layer was found to be altered; however, no lignin was present during abscission layer development. The alteration of pectic materials, coupled with breakdown of cell wall components, resulted in the collapse of cells of the abscission layer just prior to separation. Auxin delayed abscission and also delayed the initial increase in calcium, the movement of calcium from the abscission zone to the petiole, and the decrease in dry weight.     

Anatomical Changes and Immunolocalization of Cellulase during Abscission as Observed on Nitrocellulose Tissue Prints

A fundamental event in abscission is the breakdown of cell wall material in a discrete zone of cells known as the separation layer. Three dimensional images produced by viewing tissue prints of abscission zones on nitrocellulose (NC) membranes with incident illumination showed changes in the tissue integrity taking place in the separation layer as the process of abscission proceeded. The cell softening which occurs due to the dissolution of the cell wall appeared in the tissue prints as a diffuse line at the anatomical transition between the pulvinus and petiole and was easily observed on NC tissue prints of either longitudinal or serial cross-sections through abscission zones. In bean leaf abscission the dissolution of cell walls has been correlated with the appearance of a form of cellulase with an isoelectric point of pH 9.5. Antibodies specific for this enzyme were used to study the localization of 9.5 cellulase in the distal abscission zone of Phaseolus vulgaris L., cv Red Kidney after tissue printing on NC. It was found that 9.5 cellulase was localized in the separation layer but also occurred in the vascular tissue of the adjacent pulvinus. No antibody binding was observed in nonabscising tissue or preimmune controls. These results confirm previous biochemical studies and demonstrate that immunostaining of nitrocellulose tissue prints is a fast and reliable method to localize proteins or enzymes in plant tissue.     

广西蕨类植物新记录属——白桫椤属

报道了广西蕨类植物一新记录属——白桫椤属。该属植物以茎干直立,乔木状;叶大型,簇生于茎干顶端,叶柄平滑、有疣突或皮刺,基部鳞片的细胞一式;叶背灰白色;叶脉分叉,2～3叉;无囊群盖等为主要特征。目前该属植物在广西首次记录到白桫椤。根据原始文献及广西的标本对该种进行了描述,并提供了墨线图。     

A Role for the Stele in Intertissue Signaling in the Initiation of Abscission in Bean Leaves (Phaseolus vulgaris L.)

A combination of microdissection and viscometric endo-[beta]-1,4-glucanhydrolase assays was used to investigate if the early appearance of the abscission-related isoelectric point-9.5 endo-[beta]-1,4-glucanhydrolase in the stele of the pulvinus and abscission zone of the foliar abscission zone of Phaseolus vulgaris L. prior to cell separation (reported by E. del Campillo, P.D. Reid, R. Sexton, L.N.Lewis [1990] Plant Cell 2: 245-254) indicates that the vascular tissue of this region has a specific role in abscission. We find that no endo-[beta]-1,4-glucanhydrolase activity or cell separation is detectable in the abscission zone cortex if the abscission zone cortex is separated from the stele tissue. If the stele is separated from the abscission zone cortex after a lag period but again before any endo-[beta]-1,4-glucanhydrolase activity is present in the abscission zone cortex, then the enzyme is produced in the cortex and abscission ensues. We conclude that the cortex of the abscission zone is able to abscind independently of the vascular tissue only after the vascular tissue has begun to respond to abscission-promoting signals. We suggest that ethylene promotes formation of an abscission-permitting signal in the stele of the abscission zone and pulvinus, and that this signal is an essential elicitor for the synthesis of cell separation enzymes in the target cells of the abscission zone cortex.     

Polyamine-induced modulation of genes involved in ethylene biosynthesis and signalling pathways and nitric oxide production during olive mature fruit abscission

After fruit ripening, many fruit-tree species undergo massive natural fruit abscission. Olive (Olea europaea L.) is a stone-fruit with cultivars such as Picual (PIC) and Arbequina (ARB) which differ in mature fruit abscission potential. Ethylene (ET) is associated with abscission, but its role during mature fruit abscission remains largely uncharacterized. The present study investigates the possible roles of ET and polyamine (PA) during mature fruit abscission by modulating genes involved in the ET signalling and biosynthesis pathways in the abscission zone (AZ) of both cultivars. Five ET-related genes (OeACS2, OeACO2, OeCTR1, OeERS1, and OeEIL2) were isolated in the AZ and adjacent cells (AZ-AC), and their expression in various olive organs and during mature fruit abscission, in relation to interactions between ET and PA and the expression induction of these genes, was determined. OeACS2, OeACO2, and OeEIL2 were found to be the only genes that were up-regulated in association with mature fruit abscission. Using the inhibition of ET and PA biosynthesis, it is demonstrated that OeACS2 and OeEIL2 expression are under the negative control of PA while ET induces their expression in AZ-AC. Furthermore, mature fruit abscission depressed nitric oxide (NO) production present mainly in the epidermal cells and xylem of the AZ. Also, NO production was differentially responsive to ET, PA, and different inhibitors. Taken together, the results indicate that PA-dependent ET signalling and biosynthesis pathways participate, at least partially, during mature fruit abscission, and that endogenous NO and 1-aminocyclopropane-1-carboxylic acid maintain an inverse correlation, suggesting an antagonistic action of NO and ET in abscission signalling.     

ABSCISSION AND THE AXILLARY FROND IN SPIRODELA OLIGORHIZA (LEMNACEAE)

Two abscission zones are present in fronds of Spirodela oligorhiza. In one zone the abscission layer separates the daughter frond and its connecting stalk from the mother frond. The second abscission layer separates the daughter frond from the connecting stalk. An axillary frond is asymmetrically positioned at the base of each daughter frond where it joins the mother frond. In right-handed physiological clones the axillary frond is to the right of the connecting stalk and in left-handed clones, to the left of the stalk. When a daughter frond abscises it leaves behind its axillary frond in the pocket of the mother frond. Histological features of abscission and treatments that induce abscission in Spirodela oligorhiza are described.