Flavanol glucosides from rhubarb and Rhaphiolepis umbellata

Investigations of rhubarb and the bark of Rhaphiolepis umbellata led to the isolation of new flavan-3-ol glucosides. Their structures were elucidated on the basis of ¹H and ¹³C NMR analysis hydrolytic studies as (+)-catechin 5-O-β-d-glucopyranoside and (?)-catechin 7-O-β-d-glucopyranoside.     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996     

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3 from Sanguisorba officinalis

7-O-Galloyl-(+)-catechin and 3-O-galloylprocyanidin B-3, along with gambiriins A-1 and B-3 and four polygalloylglucoses, have been isolated fro     

Maize plants prime anti-herbivore responses by the memorizing and recalling of airborne information in their genome

Intact maize plants prime for defensive action against herbivory in response to herbivore-induced plant volatiles (HIPVs) emitted from caterpillar-infested conspecific plants. The recent research showed that the primed defense in receiver plants that had been exposed to HIPVs was maintained for at least 5 d after exposure. Herbivory triggered the receiver plants to enhance the expression of a defense gene for trypsin inhibitor (TI). At the upstream sequence of a TI gene, non-methylated cytosine residues were observed in the genome of HIPV-exposed plants more frequently than in that of healthy plant volatile-exposed plants. These findings provide an innovative mechanism for the memory of HIPV-mediated habituation for plant defense. This mechanism and further innovations for priming of defenses via plant communications will contribute to the development of plant volatile-based pest management methods in agriculture and horticulture.     

Inspection of frass ejection could decrease the risk of white-spotted longicorn beetle Anoplophora malasiaca (Coleoptera: Cerambycidae) infestation of Japanese pine bonsais to negligible levels

Applied Entomology and Zoology - To evaluate the infection risk of Anoplophora malasiaca (Thomson) (Coleoptera: Cerambycidae) in two species of Japanese pine bonsais (Japanese black pine and...     

Formation of unidentified nitrogen in plants: an implication for a novel nitrogen metabolism

Plants take up inorganic nitrogen and store it unchanged or convert it to organic forms. The nitrogen in such organic compounds is stoichiometrically recoverable by the Kjeldahl method. The sum of inorganic nitrogen and Kjeldahl nitrogen has long been known to equal the total nitrogen in plants. However, in our attempt to study the mechanism of nitrogen dioxide (NO₂) metabolism, we unexpectedly discovered that about one-third of the total nitrogen derived from ¹⁵N-labeled NO₂ taken up by Arabidopsis thaliana (L.) Heynh. plants was converted to neither inorganic nor Kjeldahl nitrogen, but instead to an as yet unknown nitrogen compound(s). We here refer to this nitrogen as unidentified nitrogen (UN). The generality of the formation of UN across species, nitrogen sources and cultivation environments for plants has been shown as follows. Firstly, all of the other 11 plant species studied were found to form the UN in response to fumigation with ¹⁵NO₂. Secondly, tobacco (Nicotiana tabacum L.) plants fed with ¹⁵N-nitrate appeared to form the UN. And lastly, the leaves of naturally fed vegetables, grass and roadside trees were found to possess the UN. In addition, the UN appeared to comprise a substantial proportion of total nitrogen in these plant species. Collectively, all of our present findings imply that there is a novel nitrogen mechanism for the formation of UN in plants. Based on the analyses of the exhaust gas and residue fractions of the Kjeldahl digestion of a plant sample containing the UN, probable candidates for compounds that bear the UN were deduced to be those containing the heat-labile nitrogen–oxygen functions and those recalcitrant to Kjeldahl digestion, including organic nitro and nitroso compounds. We propose UN-bearing compounds may provide a chemical basis for the mechanism of the reactive nitrogen species (RNS), and thus that cross-talk may occur between UN and RNS metabolisms in plants. A mechanism for the formation of UN-bearing compounds, in which RNS are involved as intermediates, is proposed. The important broad impact of this novel nitrogen metabolism, not only on the general physiology of plants, but also on plant substances as human and animal food, and on plants as an integral part of the global environment, is discussed.Abbreviations NO Nitric oxide - NO₂ Nitrogen dioxide - RNS Reactive nitrogen species - UN Unidentified nitrogen - TNNAT, RNNAT, INNAT and UNNAT Total, Kjeldahl, inorganic and unidentified nitrogen in naturally fed plants, respectively - TNNIT, RNNIT, INNIT and UNNIT Total, Kjeldahl, inorganic and unidentified nitrogen derived from nitrate, respectively - TNNO2, RNNO2, INNO2 and UNNO2 Total, Kjeldahl, inorganic and unidentified nitrogen derived from NO₂, respectively     

Polyamines and jasmonic acid induce plasma membrane potential variations in lima bean

Exogenous polyamines [cadaverine (Cad), putrescine (Put), spermidine (Spd) and spermine (Spm)] elicit the production of volatiles in Lima bean (Phaseolus lunatus). Among the tested PAs, Spm induces the production of some volatile terpenoids that are known to be induced by the spider mite Tetranychus urticae. Spm treatment elicits the biosynthesis of Jasmonic acid (JA), a phytohormone known to regulate the production of the volatile terpenoids. The treatment with JA together with Spm resulted in the increased volatile emission, and predatory mites Phytoseiulus persimilis preferred JA and Spm-treated leaves over those treated with JA alone.⁵ JA and Spm treatment has no effects on polyamine oxidase (PAO) and Cu-amine oxidase (CuAO) but has a significant induction of calcium influx, ROS production, enzyme activities for NADPH-oxidase complex, superoxide dismutase, catalase, ascorbate peroxidase, glutathione reductase and glutathione peroxidase, and gene expressions except for NADPH-oxidase complex.⁵ Here, we report that a plasma membrane potential (V_m) depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd. JA perfusion did not alter V_m but the perfusion of JA and the polyamines significantly increased Cad and Put V_m depolarization. When JA was perfused with polyamines, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Key words: polyamines, lima bean, herbivore-induced volatile organic compounds, calcium and ROS signalling, jasmonic acid, quantitative gene expression, transmembrane potentialPolyamines are involved in plants’ stress responses and growth. By activating biosynthesis of nucleic acids, polyamines concern the plant growth and differentiation.^1–3 Furthermore, it has been reported that polyamines are involved in the response against environmental stress and plant disease.^1–4 We recently reported that exogenously applied polyamines ∼diamines [cadaverine (Cad), putrescine (Put)], triamine [spermidine (Spd)] and tetraamine ]spermine (Spm)]∽ induce volatile emission in Lima bean leaves.⁵ Membrane potentials (V_m) and intracellular calcium variations were also studied in Lima bean leaves after perfusion with the polyamines and with these addition of JA and here we report on these additional results.The primary candidate for intercellular signaling in higher plants is the stimulus-induced change in V_m.⁶ The plasma membrane potential (V_m), which lies in the range of −50 to −200 mV in Lima bean leaves,⁷ may be shifted either to more negative (hyperpolarization) or to more positive values (depolarization) in response to various biotic or abiotic stresses.Measurement of V_m were performed and data statistically treated as previously described (ANOVA and Tukey-Kramer’s HSD test).⁷ Perfusion with the polyamines (Fig. 1 single arrow) shows a specific response of the leaf tissues with a different V_m depolarization, depending on the polyamine. In general, a V_m depolarization was observed after polyamine perfusion with an increasing trend: Spm, Cad, Put and Spd (Fig. 1). Spm and Spd V_m depolarization values were significantly different (p < 0.05) from all other polyamines, whereas no significant difference was found between Put and Cad V_m depolarization (p = 0.435). In all cases, V_m depolarization was reversed by washing polyamine-treated leaves with a fresh buffer solution (Fig. 1 double arrow); however, a full recovery of the V_m was observed only for Put (Fig. 1). The linearization of the data from Figure 1 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines which was higher for Spd (6.0 mV min⁻¹; R = 0.96), equal for Put and Cad (4.8 mV min⁻¹; Put R = 0.95; Cad R = 0.97) and lower for Spm (3.0 mV min⁻¹; R = 0.96).Open in a separate windowFigure 1Effect of 1 mM polyamines (arrow) on the V_m of Lima bean palisade cells. Spermine (Spm) caused the lowest V_m depolarization, whereas spermidine (Spd) showed the highest values of V_m depolarization. intermediate values were found when putrescine (Put) and cadaverine (cad) were perfused. after washing the tissues with fresh buffer (double arrow) V_m was always hyperpolarized, however the initial potential was recovered only for Put, while for all other polyamines the V_m never reached the initial values. Metric bars indicate standard deviation.Perfusion with JA caused a slight and not significant (p = 0.332) V_m depolarization (Fig. 2) with respect to control. The addition of JA caused a significant increase (p < 0.01) in V_m depolarization when perfused with Cad, with respect to the sole perfusion with Cad (Fig. 1). The same was observed when JA was perfused with Put, whereas not significant differences were observed when Spm (p = 0.513) and Spd (p = 0.107) were perfused with JA (Fig. 2), with respect to the sole perfusion with Spm and Spd (Fig. 1). The linearization of the data from Figure 2 allowed to calculate the rate of V_m depolarization after perfusion of the polyamines + JA, which was higher for Cad (24.40 mV min⁻¹; R = 0.99), almost equal for Put and Spd (Put: 14.21 mV min⁻¹, R = 0.99; Spd: 13.49 mV min⁻¹, R = 0.99) and lower for Spm (1.34 mV min⁻¹; R = 0.93). For JA the rate of V_m depolarization was 0.19 mV min⁻¹ (R = 0.96). With the addition of JA, a negative correlation was found between V_m depolarization and the number of amino group of the polyamines tested.Open in a separate windowFigure 2Effect of 1 mM polyamines + 0.1 mMJA (arrow) on the V_m of Lima bean palisade cells. the perfusion with Ja did not cause any variation in the V_m. addition of JA to Spm and Spd caused the same V_m depolarization observed in the absence of JA, whereas when JA was added to Put and Cad a stronger and significantly different V_m depolarization was observed. even in this case washing the tissues with fresh buffer (double arrow) caused a V_m hyperpolarized, however in this case Spd reached V_m values significantly more negative that the initial V_m. Metric bars indicate standard deviation. For abbreviations see Figure 1.Since ion fluxes through channels directly influence V_m, it seems reasonable to assume that molecules able to act on channel activity might be considered as important factors inducing electrical signals. Among the various channels, calcium and potassium channels are predominantly involved in cell signaling.⁸ In the present study, rapid and reversible V_m depolarization observed upon perfusion of Lima bean mesophyll cells with polyamines was found to be significantly increased when JA was added to Cad and Put. The reversibility of the V_m may be linked to the overall physico-chemical amphiphilic properties of polyamines, probably depending on non covalent interaction with plasma membrane molecules, as polyamines occur in plants in free form, bound electrostatically to negatively charged molecules, and conjugated to small molecules and proteins.⁹ Liu et al.¹⁰ showed that Spm, Spd, Cad and Put strongly inhibited opening and closing of stomata in Vicia faba, suggesting that polyamines target inward potassium channels in guard cells and modulate stomatal movements, so providing a link between abiotic stress, polyamine levels and stomatal regulation. Moreover, the transport of polyamines across the plasma membrane of plant cells is energy-dependent and calcium is involved in the uptake mechanism.^1,11 Both mechanisms can be correlated to the observed V_m depolarization, and the positive correlation between intracellular Ca²⁺ concentration⁵ and V_m depolarizing activity of polyamines confirms the involvement of Ca²⁺ during polyamine uptake.¹¹     

Characterization of Mesorhizobium loti L-rhamnose isomerase and its application to L-talose production

The L-rhamnose isomerase gene (rhi) of Mesorhizobium loti was cloned and expressed in Escherichia coli, and then characterized. The enzyme exhibited activity with respect to various aldoses, including D-allose and L-talose. Application of it in L-talose production from galactitol was achieved by a two-step reaction, indicating that it can be utilized in the large-scale production of L-talose.     

Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa

The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.     

Seven quinic acid gallates from quercus stenophylla

A chemical investigation of the bark of Quercus stenophylla has led to the isolation and characterization of all of the possible structural isomers of quinic acid gallates; 3-O-, 4-O-, 5-O-, 3,4-di-O-, 3,5-di-O-, 4,5-di-O- and 3,4,5-tri-O-galloylquinic acids. Evidence for the structures of these compounds was obtained from analysis of the ¹H and ¹³C NMR spectra, and hydrolytic studies.