Comparative fruit development in some Euphorbiaceae and Phyllanthaceae

Euphorbiaceae s. str. and Phyllanthaceae were earlier components of Euphorbiaceae. This separation was mainly based on molecular data and also on morphological characteristics. Nevertheless, the structure and development of fruits are poorly investigated in these families and considering this, fruits (pericarp and seed) of Euphorbia hyssopifolia L., Croton glandulosus L. (Euphorbiaceae), Phyllanthus niruri L. and Phyllanthus tenellus Roxb. (Phyllanthaceae) were structurally studied to see in what respects they are similar or different. The Euphorbiaceae schizocarps present two meristems (adaxial and subadaxial) in the ovary wall, but the adaxial meristem is entirely lacking in Phyllanthus fruits. The seeds have an exotegmen with Malpighian palisade cells in Euphorbiaceae species and a short palisade in Phyllanthus. Some special structural features, such as nucellar beak, anatropous ovules and schizocarp fruits were found in all the species and constitute a unique combination. From the present results, it appears that there is no significant difference in fruit and seed development between the studied Euphorbiaceae and Phyllanthaceae.     

Carrageenans from <Emphasis Type="Italic">Sarcothalia crispata</Emphasis> and <Emphasis Type="Italic">Gigartina skottsbergii</Emphasis>: Structural Analysis and Interpolyelectrolyte Complex Formation for Drug Controlled Release

The aims of the present study were to characterize for the first time the carrageenan extracted from cystocarpic stage of S. crispata collected in the Patagonian coast of Argentina, and to prepare interpolyelectrolytic complexes (IPECs) between the polysaccharide extracted from cystocarpic stage of Sarcothalia crispata and Gigartina skottsbergii thalli, and basic butylated methacrylate copolymer (Eudragit E), in order to test their potential for the controlled release of ibuprofen as model drug. The structural determination revealed that the polysaccharides extracted from S. crispata and G. skottsbergii were mainly constituted by κ-carrageenan, particularly in the case of G. skottsbergii; however, significant amounts of ι- and ν-carrageenan were also detected in both polygalactans. The differences in diad composition and possibly in their distribution along the polysaccharide chain of both carrageenans would favor a different arrangement in the resulting IPEC structure. The smaller pores observed by scanning electron microscopy in the IPEC of S. crispata suggest that the kinks in the polysaccharide backbone are evenly distributed, resulting in a slower ibuprofen release compared to the IPEC of G. skottsbergii.     

In vivo regulatory phosphorylation of novel phosphoenolpyruvate carboxylase isoforms in endosperm of developing castor oil seeds

Our previous research characterized two phosphoenolpyruvate (PEP) carboxylase (PEPC) isoforms (PEPC1 and PEPC2) from developing castor oil seeds (COS). The association of a shared 107-kD subunit (p107) with an immunologically unrelated bacterial PEPC-type 64-kD polypeptide (p64) leads to marked physical and kinetic differences between the PEPC1 p107 homotetramer and PEPC2 p107/p64 heterooctamer. Here, we describe the production of antiphosphorylation site-specific antibodies to the conserved p107 N-terminal serine-6 phosphorylation site. Immunoblotting established that the serine-6 of p107 is phosphorylated in COS PEPC1 and PEPC2. This phosphorylation was reversed in vitro following incubation of clarified COS extracts or purified PEPC1 or PEPC2 with mammalian protein phosphatase type 2A and is not involved in a potential PEPC1 and PEPC2 interconversion. Similar to other plant PEPCs examined to date, p107 phosphorylation increased PEPC1 activity at pH 7.3 by decreasing its K(m)(PEP) and sensitivity to L-malate inhibition, while enhancing glucose-6-P activation. By contrast, p107 phosphorylation increased PEPC2's K(m)(PEP) and sensitivity to malate, glutamic acid, and aspartic acid inhibition. Phosphorylation of p107 was promoted during COS development (coincident with a >5-fold increase in the I(50) [malate] value for total PEPC activity in desalted extracts) but disappeared during COS desiccation. The p107 of stage VII COS became fully dephosphorylated in planta 48 h following excision of COS pods or following 72 h of dark treatment of intact plants. The in vivo phosphorylation status of p107 appears to be modulated by photosynthate recently translocated from source leaves into developing COS.     

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).     

Purification and characterization of two cold-adapted extracellular tannin acyl hydrolases from an Antarctic strain <Emphasis Type="Italic">Verticillium</Emphasis> sp. P9

Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26% carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg²⁺and Br⁻ ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn²⁺, Cu²⁺, K⁺, Cd²⁺, Ag⁺, Fe³⁺, Mn²⁺, Co²⁺, Hg²⁺, Pb²⁺ and Sn²⁺), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate. Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E _a of these reactions and temperature dependence (at 0–30°C) of k _cat, k _cat/K _m, ΔG*, ΔH* and ΔS* for both the enzymes and substrates were determined. The k _cat and k _cat/K _m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II.     

Effects of isocoumarins isolated from Paepalanthus bromelioides on mitochondria: uncoupling, and induction/inhibition of mitochondrial permeability transition

Isolated mitochondria may undergo uncoupling, and in presence of Ca(2+) at different conditions, a mitochondrial permeability transition (MPT) linked to protein thiol oxidation, and demonstrated by CsA-sensitive mitochondrial swelling; these processes may cause cell death either by necrosis or by apoptosis. Isocoumarins isolated from the Brazilian plant Paepalanthus bromelioides (Eriocaulaceae) paepalantine (9,10-dihydroxy-5,7-dimethoxy-1H-naptho(2,3c)pyran-1-one), 8,8'-paepalantine dimer, and vioxanthin were assayed at 1-50 microM on isolated rat liver mitochondria, for respiration, MPT, protein thiol oxidation, and interaction with the mitochondrial membrane using 1,6-diphenyl-1,3,5-hexatriene (DPH). The isocoumarins did not significantly affect state 3 respiration of succinate-energized mitochondria; they did however, stimulate 4 respiration, indicating mitochondrial uncoupling. Induction of MPT and protein thiol oxidation were assessed in succinate-energized mitochondria exposed to 10 microM Ca(2+); inhibition of these processes was assessed in non-energized organelles in the presence of 300 microM t-butyl hydroperoxide plus 500 microM Ca(2+). Only paepalantine was an effective MPT/protein thiol oxidation inducer, also releasing cytochrome c from mitochondria; the protein thiol oxidation, unlike mitochondrial swelling, was neither inhibited by CsA nor dependent on the presence of Ca(2+). Vioxanthin was an effective inhibitor of MPT/protein thiol oxidation. All isocoumarins inserted deeply into the mitochondrial membrane, but only paepalantine dimer and vioxantin decreased the membrane's fluidity. A direct reaction with mitochondrial membrane protein thiols, involving an oxidation of these groups, is proposed to account for MPT induction by paepalantine, while a restriction of oxidation of these same thiol groups imposed by the decrease of membrane fluidity, is proposed to account for MPT inhibition by vioxanthin.     

Seed predation by birds and small mammals in semiarid Chile

We studied spatial and temporal patterns in foraging activity among diurnal birds and nocturnal mammals at a semiarid site in northern Chile using artificial foraging trays. Small mammals foraged more extensively under shrubs than in open microhabitats, but birds showed no such selection. Moreover, avian foraging was more extensive than that by small mammals in all seasons and both microhabitats. Avian foraging was highly seasonal, as many birds at our site migrate to the Andean prepuna or to Patagonia during the austral summer. Birds have tended to be overshadowed by small mammals and ants in studies of granivory, but this study suggests that their importance may be underestimated in some systems.     

Profiling phosphoproteins of yeast mitochondria reveals a role of phosphorylation in assembly of the ATP synthase

Mitochondria are crucial for numerous cellular processes, yet the regulation of mitochondrial functions is only understood in part. Recent studies indicated that the number of mitochondrial phosphoproteins is higher than expected; however, the effect of reversible phosphorylation on mitochondrial structure and function has only been defined in a few cases. It is thus crucial to determine authentic protein phosphorylation sites from highly purified mitochondria in a genetically tractable organism. The yeast Saccharomyces cerevisiae is a major model organism for the analysis of mitochondrial functions. We isolated highly pure yeast mitochondria and performed a systematic analysis of phosphorylation sites by a combination of different enrichment strategies and mass spectrometry. We identified 80 phosphorylation sites in 48 different proteins. These mitochondrial phosphoproteins are involved in critical mitochondrial functions, including energy metabolism, protein biogenesis, fatty acid metabolism, metabolite transport, and redox regulation. By combining yeast genetics and in vitro biochemical analysis, we found that phosphorylation of a serine residue in subunit g (Atp20) regulates dimerization of the mitochondrial ATP synthase. The authentic phosphoproteome of yeast mitochondria will represent a rich source to uncover novel roles of reversible protein phosphorylation.     

LPS-induced galectin-3 oligomerization results in enhancement of neutrophil activation

Galectin-3 (Gal 3) is a glycan-binding protein that can be secreted by activated macrophages and mast cells at inflammation sites and plays an important role in inflammatory diseases caused by Bacteria and their products, such as lipopolysaccharides (LPS). Although it is well established that Gal 3 can interact with LPS, the pathophysiological importance of LPS/Gal 3 interactions is not fully understood. Data presented herein demonstrate for the first time that the interaction of Gal 3, either via its carbohydrate binding C-terminal domain or via its N-terminal part, with LPS from different bacterial strains, enhances the LPS-mediated neutrophil activation in vitro. Gal 3 allowed low LPS concentrations (1 μg/mL without serum, 1 ng/mL with serum) to upregulate CD11b expression and reactive oxygen species (ROS) generation on human neutrophils in vitro and drastically enhanced the binding efficiency of LPS to the neutrophil surface. These effects required LPS preincubation with Gal 3, before neutrophil stimulation and involved specific Gal 3/LPS interaction. A C-terminal Gal-3 fragment, which retains the lectin domain but lacks the N-terminal part, was still able to bind both to Escherichia coli LPS and to neutrophils, but had lost the ability to enhance neutrophil response to LPS. This result emphasizes the importance of an N-terminus-mediated Gal 3 oligomerization induced by its interaction with LPS. Finally we demonstrated that Balb/C mice were more susceptible to LPS-mediated shock when LPS was pretreated with Gal 3. Altogether, these results suggest that multimeric interactions between Gal 3 oligomers and LPS potentiate its pro-inflammatory effects on neutrophils.     

PHYSIOLOGICAL SNAPSHOTS REFLECT ECOLOGICAL PERFORMANCE OF THE SEA PALM,POSTELSIA PALMAEFORMIS (PHAEOPHYCAEA) ACROSS INTERTIDAL ELEVATION AND EXPOSURE GRADIENTS1

Postelsia palmaeformis Ruprecht is an intertidal kelp found only on very wave‐exposed rocky shores of the northeast Pacific. In areas dominated by mussels, Postelsia depends on wave‐induced disturbances to complete its life‐history cycle. Postelsia also recruits where mussels are absent, but not at less wave‐exposed shores. Thus, physical conditions related to wave exposure limit its horizontal distribution. It is not clear what limits the vertical distribution of Postelsia. We investigated factors contributing to Postelsia's limited distribution using transplant experiments, demographic monitoring, and field fluorometry to evaluate growth and performance across gradients of tidal elevation and wave exposure. Survivorship and growth were sharply reduced at upper and wave‐protected edges relative to mid‐level, wave‐exposed sporophytes. Reproductive output was reduced at upper and lower levels, and growth but not survivorship was lower at the lower level. Effects were independent of population of origin and were a manifestation of the environment. Maximum electron transport rates (ETR_m), light saturation parameters (E_k), and maximum quantum yields (ΔF/F_m) provided insight into physiological dynamics; all were lowest at the high edge, but increased when desiccation stress was alleviated by a mock sea‐spray treatment. The ETR_m and E_k values of low sporophytes were not as high as the values for mid‐sporophytes, despite higher or equivalent nitrogen content, chl a, and absorptance, suggesting a trade‐off between light‐capturing and carbon‐fixation capacity. Physiological limitations at upper and lower levels and deleterious desiccation effects at wave‐protected sites prevent establishment, thus constraining Postelsia to a mid‐zone, wave‐exposed distribution. Physical conditions related to wave exposure may limit the horizontal distribution of Postelsia because this kelp is also found in areas where mussels are lacking but not on less wave‐exposed shores.