Studies on the Components of Mammalian Urine

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichiacoli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5′-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.     

Negatively Correlated Cross-resistance and Synergism between Phosphoramidates and Phosphorothiolates in Their Fungicidal Actions on Rice Blast Fungi

Alkyl aryl esters, dialkyl esters and diaryl esters of N-methyl-N-phenylphosphoramidic acid were synthesized and tested for cross-resistance and joint action with phosphorothiolate (PTL) fungicides using rice blast fungi, Pyricularia oryzae. Negative correlation in crossresistance was found between PTL fungicides and most of the amidates. Synergism in the fungicidal action was also found between them for wild types of the fungi, when tested by crossing filter paper strips impregnated with fungicides on agar plates inoculated uniformly with the test fungi.     

Xyloglucan oligosaccharides with at least two α-d-xylose residues act as acceptor substrates for xyloglucan endotransglycosylase and promote the depolymerisation of xyloglucan

A xyloglucan-derived pentasaccharide. Xyl₂-Glc₃, was shown by viscometry to promote the depolymerisation of xyloglucan by enzyme extracts from bean ( Phaseolus vulgaris L. cv. Canadian Wonder) leaves and pea ( Pisum sativum L. cv. Alaska) stems. Xyl₂-Glc₃ was also shown by a radiochemical assay to act as an acceptor substrate for xyloglucan endotransglycosylase activity (XET: EC 2.4.1.—) present in the same extracts. In both these assays, a heptasaccharide (Xyl₃-Glc₄) was more effective than Xyl₂-Glc₃ whereas two isomeric tetrasaccharides (Xyl₁-Glc₃) were essentially ineffective. The agreement in the structural requirements of the two assays suggests that they share a common basis; we therefore propose that the oligosaccharide-sensitive enzyme that depolymerises xyloglucan is XET rather than cellulase (EC 3.2.1.4). In the viscometric assay, the penta- and heptasaccharides would, according to our interpretation, compete with high molecular weight xyloglucan molecules as acceptor substrates for XET, leading to a decrease in the weight-average molecular weight of the xyloglucan and, therefore, to a decrease in viscosity.
Our results indicate that oligosaccharides have to possess two α- d -xylose residues in order to act as acceptor substrates for XET. The non-reducing end of a high-molecular weight xyloglucan can also act as an acceptor substrate. Therefore, it is likely that exo-hydrolysis by α- d -xylosidase would destroy the ability of a poly saccharide to act as an acceptor, even though α- d -xylosidase may remove only a single xylose residue from each polysaccharide molecule.     

香蕉果实XET cDNA的克隆及其在成熟软化的果实果皮和果肉中的表达分析

木葡聚糖内糖基转移酶(Xyloglucan endotransglycosylase,XET)通过分解细胞壁半纤维素多糖的主要成分--木葡聚糖而参与果实软化.为了阐明香蕉(Musa acuminata.Colla cv.GrandNain)果实成熟过程中的软化与细胞壁代谢酶XET基因表达模式的关系,采用RT-PCR和RACE-PCR方法,首次从成熟香蕉果实果肉中分离了编码XT基因的全长cDNA(MA-XET1,全长1 095 bp).序列分析表明,MA-XET1的5'端和3'端的非翻译区分别为66 bp和1 89bp,该片段含有一个完整的开放读码框,编码280个氨基酸,推导的MA-XET1蛋白质中存在XET蛋白的催化活性部位DEIDFEFL.Southern杂交表明,MA-XET1在香蕉基因组中由多拷贝基因编码.Northern分析显示,跃变前期的果肉中,不能检测MA-XET1基因的表达,跃变期的果实果肉中MA-XET1表达增加,跃变后期该基因表达略有减弱;在跃变前期的果实果皮中,MA-XET1的积累较低,跃变期的果实果皮中积累大幅增加,而后迅速下降.Propylene(丙烯,乙烯的类似物)处理降低香蕉果实果皮和果肉的硬度,而且propylene促进MA-XET1在果皮和果肉中的积累.这些结果表明,MA-XET1参与香蕉果实成熟过程中的果皮和果肉软化,并且,MA-XET1的表达在转录水平上受乙烯调控.     

Implication of persimmon fruit hemicellulose metabolism in the softening process. Importance of xyloglucan endotransglycosylase

Hemicellulosic polysaccharides from persimmon fruit ( Diospyros kaki L.) pericarp were extracted from depectinated cell walls with 0.5, 1 and 4 M KOH at different stages of development: (I) maximal growth corresponding to the first sigmoidal growth phase; (II) cessation of growth corresponding to the lag between the first and the second sigmoidal phases; (III) maximal growth corresponding to the second sigmoidal phase; and (IV) cessation of growth when the fruit had reached its maximum size and the change in colour (green to red) had taken place. During fruit development the amount of total hemicelluloses per unit dry mass cell wall decreased twofold. Xyloglucan was present in the three hemicellulosic fractions, and also decreased with fruit age, although its amount relative to other hemicelluloses increased. The amount of xyloglucan was especially high in the hemicelluloses extracted with 4 M KOH, representing more than 50% at stages III and IV. The average molecular mass of xyloglucan increased from stage I through stage II (0.5 M hemicellulosic fraction) or through stage III (I and 4 M hemicellulosic fractions) and decreased after that. The xyloglucan endotransglycosylase (XET: EC 2.4.1.-) activity was measured as the incorporation of [³H]XXXGol (reduced xyloglucan heptasaccharide labelled at position 1 of the glucitol moiety) into partially purified persimmon fruit xyloglucan. XET specific activity increased greatly between stages I and II. The importance of this enzyme during fruit ripening is discussed.     

Characterization of two partially purified xyloglucan endotransglycosylases from parsley (<Emphasis Type="Italic">Petroselinum crispum</Emphasis>) roots

Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from the protein mixture obtained from parsley roots and partially characterized. Both forms were glycoproteins differing in their specific activities but other features were almost the same. Activity and stability of both enzymes in broad pH region were observed with two pH optima, one at acidic pH (5.8) and the second one at basic pH (8.8). The enzymes behaved as typical transglycosylases since no activity was observed in the absence of xyloglucan oligosaccharides in the viscometric assay. Small hetero-transglycosylating activities were observed when hydroxyethyl-or carboxymethyl-celluloses instead of xyloglucan as donor substrate were used as well as when cello-oligosaccharides instead of xyloglucan oligosaccharides were used as the acceptor substrate.     

The cell wall-modifying xyloglucan endotransglycosylase/hydrolase LeXTH1 is expressed during the defence reaction of tomato against the plant parasite Cuscuta reflexa

A suppressive subtractive hybridization technique was used to identify genes, which were induced during the early phases of the interaction between dodder (Cuscuta reflexa), a phanerogamic parasite, and its incompatible host plant tomato. One of the identified genes encodes a tomato xyloglucan endotransglycosylase/hydrolase (XTH)--an enzyme involved in cell wall elongation and restructuring. The corresponding LeXTH1 mRNA accumulated 6 h after attachment of the parasite. In contrast, wounding did not influence the expression level. Subsequent to LeXTH1 mRNA accumulation, an increase in XTH activity at the infection sites as well as in adjacent tissues was observed. The effect of IAA on LeXTH1 expression was analyzed because the concentration of this phytohormone is known to increase in the tomato tissue during the interaction with the parasite. LeXTH1 mRNA accumulation was in fact induced by external application of auxin. However, in the auxin-insensitive tomato mutant diageotropica, Cuscuta induced LeXTH1-mRNA accumulated with a time course similar to wild type tomato. Thus, auxin appears not to be an essential signal for infection-induced LeXTH1 activation. Our data suggest a role for xyloglucan transglycosylation in defence reactions associated with the incompatible tomato- Cuscuta interaction.     

Molecular genetic analysis of brassinosteroid action

Recent applications of molecular techniques to the study of brassinosteroid action have enhanced our understanding of these unique plant growth regulators. The cloning of genes regulated by brassinosteroids has revealed novel information on the control of gene expression by plant steroids and has extended our knowledge of brassinosteroid-promoted cell expansion. The analysis of brassinosteroid-deficient and brassinosteroid-insensitive mutants has implicated these growth regulators in a number of essential developmental programs including organ elongation, leaf development, photomorphogenesis, fertility, apical dominance and vascular differentiation.     

Role of transglycosylation in the integration of xyloglucan into the growing plant cell wall in vivo

Abstract

Xyloglucan endotransglycosylase (XET) activity is widespread in plant cell walls, but its action on xyloglucan in vivo has been difficult to prove because the reaction products are not expected to differ chemically from the reactants. By feeding of cultured Rosa cells with [¹³C]glucose and [³H]arabinose followed by [¹²-C]glucose, and isopyenic centrifugation of the extracted xyloglucan in caesium trifluoroacetate, we have obtained evidence for the annealing of segments of newly-secreted xyloglucan to xyloglucan chains that were already present in the cell wall. This is the first evidence for interpolymeric transglycosylation of xyloglucan in vivo.     

Effects of tannery waste on growth dynamics and metal uptake in Salix alba L

Abstract

This paper aims at providing a preliminary study of the effects of different doses of tannery waste containing high amounts of organic substances, mineral elements and heavy metals on growth dynamics and metal accumulation in willow plants. Scions of Salix alba L., clone S61-02, were grown in the open near Pisa (Italy), in pots filled with a natural soil. Willows were pruned for a unique shoot and cultivated with three different treatments: non-amended soil, soil amended with 150 or 300 g of tannery waste. Non-destructive measures of plant height and leaf area were performed during one growth season. Determination of biomass, and chemical analyses of the minerals were performed at the end of the experiment. The waste-treated plants increased all growth parameters significantly more than the non-treated ones. The concentration of the analysed elements (N, P, K, Ca, Mg, S, B, Fe, Mn, Cu, Zn, Cr) changed significantly between treatments and organs. The results can be useful for developing phytostabilization and/or phytoextraction strategies of tannery waste using S. alba.     

Restructuring of wall-bound xyloglucan by transglycosylation in living plant cells

Xyloglucan endotransglycosylases (XETs) cleave and then re-join xyloglucan chains and may thus contribute to both wall-assembly and wall-loosening. The present experiments demonstrate the simultaneous occurrence in vivo of two types of interpolymeric transglycosylation: "integrational" (in which a newly secreted xyloglucan reacts with a previously wall-bound one) and "restructuring" (in which one previously wall-bound xyloglucan reacts with another). Xyloglucans synthesised by cultured rose (Rosa sp.) cells in "heavy" or "light" media (with [13C,2H]glucose or [12C,1H]glucose, respectively) had buoyant densities of 1.643 and 1.585 g ml-1, respectively, estimated by isopycnic centrifugation in caesium trifluoroacetate. To detect transglycosylation, we shifted heavy rose cells into light medium, then supplied a 2-h pulse of L-[1-3H]arabinose. Light [3H]xyloglucans were thus secreted into heavy, non-radioactive walls and chased by light, non-radioactive xyloglucans. At 2 h after the start of radiolabelling, the (neutral) [3H]xyloglucans were on average 29% heavy, indicating molecular grafting during integrational transglycosylation. The [3H]xyloglucans then gradually increased in density until, by 11 h, they were 38% heavy. This density increase suggests that restructuring transglycosylation reactions occurred between the now wall-bound [3H]xyloglucan and other (mainly older, i.e. heavy) wall-bound non-radioactive xyloglucans. Brefeldin A (BFA), which blocked xyloglucan secretion, did not prevent the increase in density of wall-bound [3H]xyloglucan (2-11 h). This confirms that restructuring transglycosylation occurred between pairs of previously wall-bound xyloglucans. After 7 d in BFA, the 3H was in hybrid xyloglucans in which on average 55% of the molecule was heavy. Exogenous xyloglucan oligosaccharides (competing acceptor substrates for XETs) did not affect integrational transglycosylation whereas they inhibited restructuring transglycosylation. Possible reasons for this difference are discussed. This is the first experimental evidence for restructuring transglycosylation in vivo. We argue that both integrational and restructuring transglycosylation can contribute to both wall-assembly and -loosening.     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

桑枝提取物及抗炎作用研究

桑支（Ramulus mori)乙醇提取物经不同有机溶剂萃取,浓缩得到石油醚提取物（I）,乙酸乙酯提取物（Ⅱ）和正丁醇提取物（Ⅲ）三部分,分别进行定性分析。并灌胃给药进行抗炎实验,结果显示,I和Ⅲ的0.30g/kg组对二甲苯致小鼠耳肿胀有明显的抑制作用,I的0.15g/kg组有明显的抑制毛细血管通透性的作用。     

薄荷属植物挥发性成分及药理作用研究进展

薄荷属(Mentha L.)植物广泛分布于世界各地,有着悠久的药食两用史。目前,薄荷属植物挥发性物质主要应用于医药、食品、化妆品、香料、烟草等工业。现代研究表明其挥发性成分主要含有多种单萜类化合物,药理学研究显示其具有抗氧化、抗菌、抗辐射、抗癌、降血压等生物活性。本文主要对该属植物挥发性化学成分及药理作用的研究现状进行综述,以期为进一步开发和利用该属植物提供科学依据。     

大叶盘果菊化学成分的研究

采用乙醇提取,硅胶柱层析分离和波谱方法鉴定结构,从大叶盘果菊(Prenanthes macrophylla Franch)中初步分离鉴定出8个化合物:木栓酮(friedelin,1)、木栓醇(friedelanol,2)、α-香树脂醇(α-amyrin,3)、α-香树脂醇乙酸脂(α-amyrin acetate,4)、β-香树脂醇乙酸酯(β-amyrin acetate,5)、蒲公英甾醇乙酸酯(taraxasteryl acetate,6)、β-谷甾醇(β-sitosterol,7)、二十八烷酸(octacosanoic acid,8),这些化合物均为首次从该植物中分离得到。     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

Simultaneous determination of five characteristic stilbene glycosides in root bark of Morus albus L. (Cortex Mori) using high-performance liquid chromatography

Introduction – Cortex Mori, one of the well‐known traditional Chinese herbal medicines, is derived from the root bark of Morus alba L. according to the China Pharmacopeia. Stilbene glycosides are the main components isolated from aqueous extracts of Morus alba and their content varies depending on where Cortex Mori was collected. We have established a qualitative and quantitative method based on the bioactive stilbene glycosides for control of the quality of Cortex Mori from different sources. Objective – To develop a high‐performance liquid chromatography coupled with ultraviolet absorption detection for simultaneous quantitative determination of five major characteristic stilbene glycosides in 34 samples of the root bark of Morus alba L. (Cortex Mori) from different sources. Methodology – The analysis was performed on an ODS column using methanol‐water‐acetic acid (18: 82: 0.1, v/v/v) as the mobile phase and the peaks were monitored at 320 nm. Results – All calibration curves showed good linearity (r ≥ 0.9991) within test ranges. This method showed good repeatability for the quantification of these five components in Cortex Mori with intra‐ and inter‐day standard deviations less than 2.19% and 1.45%, respectively. Conclusion – The validated method was successfully applied to quantify the five investigated components, including a pair of cis‐trans‐isomers 1 and 2 and a pair of isomers 4 and 5 in 34 samples of Cortex Mori from different sources. Copyright © 2010 John Wiley & Sons, Ltd.     

Life in a changing world: TCH gene regulation of expression and responses to environmental signals

The Arabidopsis TCH genes were discovered as a consequence of their marked upregulation of expression in response to seemingly innocuous stimuli, such as touch. Further analyses have indicated that these genes are upregulated by a variety of diverse stimuli. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signalling pathways that enable plants to respond to changing environmental conditions. The TCH proteins include calmodulin, calmodulin-related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicate that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that the TCH proteins may collaborate in cell wall biogenesis.