Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Thiol addition to protected allyl glycosides: An improved method for the preparation of spacer-arm glycosides

A useful method for the preparation of differently functionalized sulfide spacer-arm glycosides is presented. Several protected allyl glycosides were variously elongated via a radical addition reaction with pentanethiol, methyl 3-mercaptopropionate, or 2-mercaptoethanol. The hydroxyl function of protected 3-(2-hydroxyethylthio)propyl glycosides was subsequently transformed into an azide function.     

Identification of an intracellular domain of the EGF receptor required for high-affinity binding of EGF

Although all EGF receptors in EGF receptor-expressing cells are molecularly identical, they can be subdivided in two different classes that have either a high or a low affinity for EGF. Specifically the high-affinity class is associated with filamentous actin. To determine whether the interaction of the EGF receptor with actin induces its high-affinity state, we studied EGF-binding properties of an EGF receptor mutant that lacks the actin-binding site. Interestingly, we found that cells expressing this mutant receptor still display both high- and low-affinity classes of EGF receptors, indicating that the actin-binding domain does not determine the high-affinity binding state. By further mutational analysis we identified a receptor domain, within the tyrosine kinase domain, that regulates the affinity for EGF.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Three differentially expressed S-adenosylmethionine synthetases from Catharanthus roseus: molecular and functional characterization

We describe the molecular and functional characterization of three closely related S-adenosyl-L-methionine synthetase (SAMS) isoenzymes from Catharanthus roseus (Madagascar periwinkle). The genes are differentially expressed in cell cultures during growth of the culture and after application of various stresses (elicitor, nutritional down-shift, increased NaCl). Seedlings revealed organ-specific expression and differential gene regulation after salt stress. A relationship analysis indicated that plant SAMS group in two main clusters distinguished by characteristic amino acid exchanges at specific positions, and this suggested differences in the enzyme properties or the regulation. SAMS1 and SAMS2 are of type I and SAMS3 is of type II. The properties of the isoenzymes were compared after heterologous expression of the individual enzymes, but no significant differences were detected in a) optima for temperature (37 to 45 °C) or pH (7 to 8.3); b) dependence on cations (divalent: Mg2+, Mn2+, Co2+; monovalent: K+, , Na+); c) Kms for ATP and L-methionine; d) inhibition by reaction products (S-adenosyl-L-methionine, PPi, Pi), by the reaction intermediate tripolyphosphate, and by the substrate analogues ethionine and cycloleucine; e) response to metabolites from the methyl cycle (L-homocysteine) or from related pathways (L-ornithine, putrescine, spermidine, spermine); f) native protein size (gel permeation chromatography). The results represent the first characterization of plant SAMS isoenzyme properties with individually expressed proteins. The possibility is discussed that the isoenzyme differences reflect specificities in the association with enzymes that use S-adenosyl-L-methionine.     

Site fertility and leaf nutrients of sympatric evergreen and deciduous species of Quercus in central coastal California

Leaf and soil nutrient levels interact with and may each influence the other. We hypothesize that to the extent soil fertility influences the nutritional state of trees, soil fertility should correlate with summer leaf nutrient levels, whereas to the extent that trees influence soil nutrient levels, the quality of leaf litterfall should correlate with soil fertility. We examined these correlations for five sympatric oak species (genus Quercus) in central coastal California. Soil fertility, including both nitrogen and especially phosphorus, correlated significantly with summer leaf nutrient levels. In contrast, phosphorus, but not nitrogen, in the leaf litterfall correlated positively with soil nutrients. These results suggest that soil nitrogen and phosphorus influence tree nutrient levels and that leaf phosphorus, but not leaf nitrogen, influence soil fertility under the trees. Feedback between the soil and the tree for phosphorus, but not nitrogen, is apparently significant and caused by species-specific differences in leaf quality and not by litterfall quality differences within a species. We also compared functional differences between the evergreen and deciduous oak species at our study site. There were no differences in soil nitrogen and only small differences for soil phosphorus between the phenological types. Differences in leaf nutrient concentration were much more pronounced, with the evergreen species having substantially lower levels of both nitrogen and phosphorus. Evergreen species conserved more phosphorus, but not more nitrogen, than the deciduous species, but there was no consistent relationship between retranslocation and either soil nitrogen or phosphorus. These results do not support the hypothesis that evergreenness is an adaptation to low soil fertility in this system.     

ENDOGENOUS CYTOKININS IN THREE GENERA OF MICROALGAE FROM THE CHLOROPHYTA1

Nine axenic microalgal (Chlorophyta) strains from three genera (Protococcus, Chlorella, and Scenedesmus) were analyzed for endogenous cytokinins. Cytokinin‐like activity was detected using the excised cucumber cotyledon bioassay. Five strains showed no cytokinin‐like activity and four strains, low cytokinin‐like activity. Ethanolic extracts of the microalgae containing a mixture of deuterium‐labeled standards were purified using a combined DEAE‐Sephadex octadecysilica column and immunoaffinity column based on wide‐range specific mon‐oclonal antibodies and analyzed by HPLC linked to a micromass single quadrupole mass spectrometer with an electrospray interface and a photodiode array detector. There were similar trends in cytokinin profiles for the nine microalgal strains investigated, although concentrations did vary. Both isopentenyladenine and isopentenyladenosine were detected in all nine strains. cis‐Zeatin and cis‐zeatin riboside occurred at higher concentrations than the trans isomers, whereas trans‐zeatin‐O‐glucoside and trans‐zeatin riboside‐O‐glucoside were dominant over the cis isomers. Dihydrozeatin and its conjugates were not detected in any significant amounts. The aromatic benzyladenine always occurred at higher concentrations than benzyladenosine. The topolins were well represented with all three isomers (ortho, meta, and para) being detected, with ortho‐topolin and ortho‐topolin riboside occurring at higher concentrations than the other isomers. However, for the O‐glucosides, the meta isomers (meta‐topolin‐O‐glucoside and meta‐topolin riboside‐O‐glucoside) occurred at higher concentrations than the other isomers. No N‐glucosides were detected (isopentenyladenine‐9‐glucoside, zeatin‐9‐glucoside, dihydrozeatin‐9‐glucoside, benzyladenine‐9‐glucoside, ortho‐topolin‐9‐glucoside, and meta‐topolin‐9‐glucoside). Generally, zeatin and topolin conjugates were the dominant forms of isoprenoid and aromatic cytokinins, respectively. There was no distinct trend in the proportions of isoprenoid to aromatic cytokinins.     

Enhancement of intracellular concentration and biological activity of PNA after conjugation with a cell-penetrating synthetic model peptide.

In order to evaluate the ability of the cell-penetrating alpha-helical amphipathic model peptide KLALKLALKALKAALKLA-NH(2) (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12-mer PNA 5'-GGAGCAGGAAAG-3' directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP-conjugate was studied by means of capillary electrophoresis combined with laser-induced fluorescence detection, confocal laser scanning microscopy and fluorescence-activated cell sorting. Incubation with the fluorescein-labelled PNA-peptide conjugate led to three- and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously-beating neonatal rat cardiomyocytes with the PNA-peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10- and twofold, respectively. The main reasons for the higher bioavailability of the PNA-peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re-export and resistance against influences of serum.     

The role of plant species in biomass production and response to elevated CO2 and N

In an experiment that factorially manipulated plant diversity, CO₂, and N, we quantified the effects of the presence of species on assemblage biomass over 10 time points distributed over 5 years. Thirteen of the 16 species planted had statistically significant effects on aboveground and/or belowground biomass. Species differed dramatically in their effects on biomass without any relationship between aboveground and below‐ground effects. Temporal complementarity among species in their effects seasonally, successionally, and in response to a dry summer maintained the diversity–biomass relationships over time and may be the cause behind higher diversity plots having less variation in biomass over time. The response of plant biomass to elevated N, but not CO₂, was at times entirely dependent on the presence of a single species.     

Rhodocetin antagonizes stromal tumor invasion in vitro and other alpha2beta1 integrin-mediated cell functions.

The pleiotropic effects of Calloselasma rhodostoma venom is caused by various toxins, among them kistrin and ancrod, which block platelet activation triggered by RGD-dependent integrins and the blood clotting cascade, respectively. Here, we demonstrate that rhodocetin, another component of this venom, acts as alpha2beta1 integrin inhibiting disintegrin and antagonizes important cellular responses to type I collagen. Cell adhesion, migration, and collagen lattice contraction in vitro were specifically inhibited by rhodocetin, whereas expression of collagen-degrading matrix metalloproteases was differently modulated. Moreover, cell invasion of HT1080 fibrosarcoma cells into a type I collagen matrix, but not into a fibrin gel or a basement membrane-extracted matrigel was efficiently blocked by rhodocetin. Unlike its natural ligand collagen, rhodocetin failed to cluster alpha2beta1 integrin, despite similar binding affinities. Hence, in the absence of focal adhesions cells do not attach firmly to rhodocetin and do not respond with any of alpha2beta1-triggered cell reactions, except for MMP-1 production. Therefore, this disintegrin may be a valuable tool to specifically target stromal tumor invasion and to manipulate other alpha2beta1 integrin-mediated functions, such as excessive scar contraction and fibrosis. Rhodocetin might be therapeutically useful because of its lack of interference with RGD-dependent integrins, low molecular mass, high solubility, and biochemical stability.