Positive interactions among plant species for pollinator service: assessing the ‘magnet species’ concept with invasive species

Plants with poorly attractive flowers or with little floral rewards may have inadequate pollinator service, which in turn reduces seed output. However, pollinator service of less attractive species could be enhanced when they are associated with species with highly attractive flowers (so called ‘magnet‐species’). Although several studies have reported the magnet species effect, few of them have evaluated whether this positive interaction result in an enhancement of the seed output for the beneficiary species. Here, we compared pollinator visitation rates and seed output of the invasive annual species Carduus pycnocephalus when grow associated with shrubs of the invasive Lupinus arboreus and when grow alone, and hypothesized that L. arboreus acts as a magnet species for C. pycnocephalus. Results showed that C. pycnocephalus individuals associated with L. arboreus had higher pollinator visitation rates and higher seed output than individuals growing alone. The higher visitation rates of C. pycnocephalus associated to L. arboreus were maintained after accounting for flower density, which consistently supports our hypothesis on the magnet species effect of L. arboreus. Given that both species are invasives, the facilitated pollination and reproduction of C. pycnocephalus by L. arboreus could promote its naturalization in the community, suggesting a synergistic invasional process contributing to an ‘invasional meltdown’. The magnet effect of Lupinus on Carduus found in this study seems to be one the first examples of indirect facilitative interactions via increased pollination among invasive species.     

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.     

Inactivation of Na,K-ATPase following Co(NH3)4ATP binding at a low affinity site in the protomeric enzyme unit

The Na(+)-dependent or E1 stages of the Na,K-ATPase reaction require a few micromolar ATP, but submillimolar concentrations are needed to accelerate the K(+)-dependent or E2 half of the cycle. Here we use Co(NH(3))(4)ATP as a tool to study ATP sites in Na,K-ATPase. The analogue inactivates the K(+) phosphatase activity (an E2 partial reaction) and the Na,K-ATPase activity in parallel, whereas ATP-[(3)H]ADP exchange (an E1 reaction) is affected less or not at all. Although the inactivation occurs as a consequence of low affinity Co(NH(3))(4)ATP binding (K(D) approximately 0.4-0.6 mm), we can also measure high affinity equilibrium binding of Co(NH(3))(4)[(3)H]ATP (K(D) = 0.1 micro m) to the native enzyme. Crucially, we find that covalent enzyme modification with fluorescein isothiocyanate (which blocks E1 reactions) causes little or no effect on the affinity of the binding step preceding Co(NH(3))(4)ATP inactivation and only a 20% decrease in maximal inactivation rate. This suggests that fluorescein isothiocyanate and Co(NH(3))(4)ATP bind within different enzyme pockets. The Co(NH(3))(4)ATP enzyme was solubilized with C(12)E(8) to a homogeneous population of alphabeta protomers, as verified by analytical ultracentrifugation; the solubilization did not increase the Na,K-ATPase activity of the Co(NH(3))(4)ATP enzyme with respect to parallel controls. This was contrary to the expectation for a hypothetical (alphabeta)(2) membrane dimer with a single ATP site per protomer, with or without fast dimer/protomer equilibrium in detergent solution. Besides, the solubilized alphabeta protomer could be directly inactivated by Co(NH(3))(4)ATP, to less than 10% of the control Na,K-ATPase activity. This suggests that the inactivation must follow Co(NH(3))(4)ATP binding at a low affinity site in every protomeric unit, thus still allowing ATP and ADP access to phosphorylation and high affinity ATP sites.     

Saprolegnia oliviae sp. nov. isolated from an Argentine river (Tierra del Fuego Province,Argentina)

Saprolegnia oliviae sp. nov. is described from litter (floating dead twigs, leaves and roots) in the Olivia River, Ushuaia Department, Tierra del Fuego Province (Argentina). The new species is illustrated and compared with other species of the genus. Distinguishing characteristics of S. oliviae are the production of smooth oogonia (with some lateral or terminal projections) and the absence of antheridial branches on the majority of the oogonia, but when present, they are mostly diclinous, at times oogonia are supplied with androgynous and monoclinous antheridial branches. The oogonial stalks are predominantly short and straight or long and bent, curved or many times coiled; oospores are distinctive subcentric, (1-) 15-50 (-70) per oogonium. Morphological details of the new species and its comparison with other described species are discussed here.     

Irreversible effects of calcium ions on the plasma membrane calcium pump

The calcium pump of human red cells can be irreversibly activated by preincubation of the membranes in the presence of calcium ions, with a pattern reminiscent of that produced by controlled trypsin attack. With 1 mm Ca²⁺, the activity of the basal enzyme increases three to fourfold over 30 to 60 min, to levels about half those obtained in the presence of calmodulin. On the whole, the effect occurs slowly, with a very low Ca²⁺ affinity at 37°C and is unaffected by serine-protease inhibitors. The activation caused by 1 mm Ca²⁺ is little affected by leupeptin (a thiol-protease inhibitor) and that obtained at 10 m Ca²⁺ is not inhibited. Preincubations at 0°C also lead to activation, to a level up to half that seen at 37°C, and the effect is not affected by leupeptin or antipain. No activation is observed by preincubating soluble purified Ca,Mg-ATPase in Ca²⁺-containing solutions at 37°C. Instead, calcium ions protect the detergent-solubilized enzyme from thermal inactivation, the effect being half-maximal between 10 and 20 m Ca²⁺. We conclude that the activation of the membrane-bound Ca,Mg-ATPase by Ca²⁺ should result from an irreversible conformational change in the enzyme and not from attack by a membrane-bound protease, and that this change presumably arises from the release of inhibitory particles existing in the original membrane preparations.We thank The Wellcome Trust for a research grant, the Medical Research Council for an equipment grant and the Regional Transfusion Service (Sheffield) for bank blood supplies.     

Positive feedbacks between plant invasions and fire regimes: <Emphasis Type="Italic">Teline monspessulana</Emphasis> (L.) K. Koch (Fabaceae) in central Chile

Invasive species can increase fire frequency and intensity, generating favorable conditions for their self-perpetuation. Mediterranean south-central Chile may be especially prone to the effects of invasive species on fire regimes because it is less adapted to fire and it contains a highly endemic flora. Teline monspessulana (L.) K. Koch (syn. Cytisus monspessulanus L.; Genista monspessulana (L.) L.A.S. Johnson) is an introduced shrub that forms monotypic stands or is present as an understory species in native forests as well as in forestry plantations. Dense T. monspessulana stands are completely destroyed by fire, generating the conditions for it seeds to germinate and establish an abundant regeneration, with up to 900 plants/m². We report key evidence on abundance and biomass in adult stands, and patterns of seed bank and regeneration after fire in stands of T. monspessulana around the city of Concepción, Chile. We estimated living biomass in pure stands and underneath Eucalyptus plantations. In burned areas, we assessed T. monspessulana seed bank and studied regeneration patterns. We found that T. monspessulana densities reaches 52,778 plants/ha and 8.92 ton/ha in pure stands and 34,223 plants/ha and 2.31 ton/ha underneath Eucalyptus plantations. T. monspessulana generates small caliper fuel and acts as a ladder-fuel. Large soil seed banks allow for abundant regeneration after fire, with mean densities of 877,111 plants/ha, but an overall mortality of 37.2% in the first year after the fire. The high values of regeneration compared to final densities in adult stands suggest that density-dependent mortality. Our results indicate that T. monspessulana regeneration is not only favored by fires, but also that the species creates favorable conditions for intense and continuous fires, both under pure conditions, but also associated to exotic tree plantations. To understand the implications of positive feedbacks between invaders and fire, we recommend focusing in the mechanisms by which they increases fuel accumulation and fuel flammability, and how higher fire frequency and intensity favors invasive species recruitment over native species. Comprehension of this dynamics will allow for better management and control of these invasions which have major ecological, economical and social implications.     

Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

Reduction of infarct size and postischemic inflammation from ATL-146e, a highly selective adenosine A2A receptor agonist, in reperfused canine myocardium

Adenosine and adenosine A(2A) receptor agonists have been shown to limit myocardial infarct size when given at vasodilatory doses during reperfusion. This beneficial effect is thought to be due, in part, to stimulation of adenosine A(2A) receptors on inflammatory cells. The specific aims of this study were to determine whether the anti-inflammatory and cardioprotective properties of a novel adenosine A(2A) receptor agonist, ATL-146e (ATL), alone or in combination with the phosphodiesterase IV inhibitor rolipram would occur using very low, nonvasodilating doses. In a canine model of reperfused myocardial infarction, low-dose ATL given alone reduced infarct size by 45% (P < 0.05 vs. control). When ATL was combined with a very low dose of rolipram (0.001 microg.kg(-1).min(-1)), a marked reduction in P-selectin expression and neutrophil infiltration (51% lower; P < 0.001 vs. control) was seen and the infarct size reduction (58% lower; P < 0.01 vs. control) was greater than observed with ATL (45% lower; P < 0.05) or rolipram (33% lower; P < 0.05) alone. In conclusion, a low, nonvasodilating dose of ATL, a highly selective adenosine A(2A) receptor agonist, reduced infarct size after reperfusion. Furthermore, combining ATL and the phosphodiesterase IV inhibitor rolipram reduced infarct size even more than either agent alone. Such combination therapy may be beneficial clinically by potentiating cardioprotection after coronary reperfusion at doses far below those producing vasodilatation or side effects.     

Expression and characterization of HlyX hemolysin from Leptospira interrogans serovar Copenhageni: potentiation of hemolytic activity by LipL32

The HlyX, a putative hemolysin identified from the Leptospira genomes, was cloned, expressed in Escherichia coli, purified, and its hemolytic activity was confirmed. Mouse polyclonal antiserum against the recombinant HlyX recognized HlyX-related antigens in a panel of Leptospira species extracts and it was also able to abolish the hemolytic activity of HlyX. A mixture of HlyX and LipL32, a known hemolysin from Leptospira, induced hemolysis in a synergistic way that was fully inhibited by antiserum against either protein. Moreover, sera from patients with leptospirosis also recognized the recombinant HlyX, showing that it is presented to the host immune system during Leptospira infection.     

Non-enzymatic in vitro DNA labeling and label immunoquantification

In the present work, a label immunoquantification procedure was developed in order to determine the number of markers introduced into DNA. A non-enzymatic, in vitro labeling method for introducing the p-bromobenzoyl radical (label), through transamination and acylation reactions of the cytidine nucleotides in calf thymus DNA, was carried out. Three spacer arms with different lengths were used for separating the label from the nucleotide and three labeled DNA were obtained. Anti-p-bromobenzoyl chicken IgY polyclonal antibodies were obtained. The antibodies detected the label, into three-labeled DNA, with different sensitivities, in relation to spacer arm length used. About 3-11 labels per 4 x 10(6) bases into thermally denatured DNA were immunoquantified.