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.     

Flavonoids from Flindersia australis

Dihydrokaempferol, dihydrokaempferol 3-O-rhamnoside (engeletin) and kaempferol were isolated from the stem bark of Flindersia australis. This is the first report of the occurrence of these flavonoids in Flindersia.     

Long term effects of manure, charcoal and mineral fertilization on crop production and fertility on a highly weathered Central Amazonian upland soil

Application of organic fertilizers and charcoal increase nutrient stocks in the rooting zone of crops, reduce nutrient leaching and thus improve crop production on acid and highly weathered tropical soils. In a field trial near Manaus (Brazil) 15 different amendment combinations based on equal amounts of carbon (C) applied through chicken manure (CM), compost, charcoal, and forest litter were tested during four cropping cycles with rice (Oryza sativa L.) and sorghum (Sorghum bicolor L.) in five replicates. CM amendments resulted in the highest (P < 0.05) cumulative crop yield (12.4 Mg ha⁻¹) over four seasons. Most importantly, surface soil pH, phosphorus (P), calcium (Ca), and magnesium (Mg) were significantly enhanced by CM. A single compost application produced fourfold more grain yield (P < 0.05) than plots mineral fertilized in split applications. Charcoal significantly improved plant growth and doubled grain production if fertilized with NPK in comparison to the NPK-fertilizer without charcoal (P < 0.05). The higher yields caused a significantly greater nutrient export in charcoal-amended fields, but available nutrients did not decrease to the same extent as on just mineral fertilized plots. Exchangeable soil aluminum (Al) was further reduced if mineral fertilizer was applied with charcoal (from 4.7 to 0 mg kg⁻¹). The resilience of soil organic matter (SOM) in charcoal amended plots (8 and 4% soil C loss, mineral fertilized or not fertilized, respectively) indicates the refractory nature of charcoal in comparison to SOM losses over 20 months in CM (27%), compost amended (27%), and control plots (25% loss).     

IADS, a decomposition product of DIDS activates a cation conductance in Xenopus oocytes and human erythrocytes: new compound for the diagnosis of cystic fibrosis.

DIDS (4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid) is a commonly used blocker of plasma membrane anion channels and transporters. We observed that DIDS undergoes decomposition while stored in DMSO (dimethyl sulfoxide) forming a biologically active compound. One decomposition product, called IADS, was identified and synthesized. Voltage-clamp and patch clamp experiments on Xenopus laevis oocytes and human erythrocytes revealed that IADS is able to activate a plasma membrane cation conductance in both cell types. Furthermore, we found that IADS induces hemolysis in red blood cells of healthy donors but fails to hemolyze erythrocytes of donors with cystic fibrosis. Thus, IADS stimulated activation of a cation conductance could form the basis for a novel diagnostic test of cystic fibrosis.     

Turnover of passerine birds on islands in the Aegean Sea (Greece)

Aim We wish to determine the effect of migratory status on turnover rates in island birds. Because turnover is influenced by factors other than migratory status, we also considered the influence of body size and physical characteristics of the islands inhabited on the probabilities of extinction and immigration. Location The Mediterranean islands of Delos, Astypalea, Paros, Naxos and Lesvos in the Aegean Sea, Greece. Methods The passerine birds of these islands were surveyed between 1954 and 1961 by G.E. Watson, and were resurveyed between 1988 and 1992. The effects of migratory status and body size on the probabilities of extinction and immigration were examined by G‐tests of linear trend in proportion, and analysis of variance, respectively. A combined analysis of migratory status, body size and physical characteristics of the islands was carried out using logistic regressions of the probabilities of extinction and immigration on these factors. Results Species number on each island changed little between surveys, with no island's species number changing by more than one species. Twelve population extinctions and 11 immigrations were recorded. The smallest island, Delos (6 km²), had the highest annualized relative turnover rate (1.08), while the four larger islands (96–1614 km²) had lower and mutually similar rates (0.21–0.27). Populations on higher elevation islands were less likely to go extinct. There is no evidence for an effect of body size on the probabilities of extinction or immigration. Migratory status affected extinction and immigration probabilities differently: migratory species were more likely to immigrate, but less likely to go extinct. Main conclusions The position of the Aegean islands along a major north–south flyway may account for the observed effects of migratory status. The annual passage of large numbers of migrants may, via the rescue effect, decrease the chances of extinction, while at the same time increasing the chances of colonization of unoccupied islands. The likelihood of both extinction and immigration involves a complex interaction between life‐history traits and island characteristics. The effects of migratory status will depend not only on consideration of vagility, vulnerability and stochasticity identified by previous authors, but also upon the location of the islands in relationship to migratory pathways.