Identification of novel oxidized levuglandin D2 in marine red alga and mouse tissue

In animals, the product of cyclooxygenase reacting with arachidonic acid, prostaglandin(PG)H(2), can undergo spontaneous rearrangement and nonenzymatic ring cleavage to form levuglandin(LG)E(2) and LGD(2). These LGs and their isomers are highly reactive γ-ketoaldehydes that form covalent adducts with proteins, DNA, and phosphatidylethanolamine in cells. Here, we isolated a novel oxidized LGD(2) (ox-LGD(2)) from the red alga Gracilaria edulis and determined its planar structure. Additionally, ox-LGD(2) was identified in some tissues of mice and in the lysate of phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells incubated with arachidonic acid using LC-MS/MS. These results suggest that ox-LGD(2) is a common oxidized metabolite of LGD(2). In the planar structure of ox-LGD(2), H8 and H12 of LGD(2) were dehydrogenated and the C9 aldehyde was oxidized to a carboxylic acid, which formed a lactone ring with the hydrated ketone at C11. These structural differences imply that ox-LGD(2) is less reactive with amines than LGs. Therefore, ox-LGD(2) might be considered a detoxification metabolite of LGD(2).     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n     

Identification of C4 responsive genes in the facultative C4 plant Hydrilla verticillata

The aquatic monocot Hydrilla verticillata (L.f.) Royle is a well-documented facultative C₄ NADP-malic enzyme species in which the C₄ and Calvin cycles operate in the same cell with the specific carboxylases confined to the cytosol and chloroplast, respectively. Several key components had already been characterized at the molecular level, thus the purpose of this study was to begin to identify other, less obvious, elements that may be necessary for a functional single-cell C₄ system. Using differential display, mRNA populations from C₃ and C₄ H. verticillata leaves were screened and expression profiles compared. From this study, 65 clones were isolated and subjected to a customized macroarray analysis; 25 clones were found to be upregulated in C₄ leaves. Northern and semi-quantitative RT-PCR analyses were used for confirmation. From these screenings, 13 C₄ upregulated genes were identified. Among these one encoded a previously recognized C₄ phosphoenolpyruvate carboxylase, and two encoded distinct pyruvate orthophosphate dikinase isoforms, new findings for H. verticillata. Genes that encode a transporter, an aminotransferase and two chaperonins were also upregulated. Twelve false positives, mostly housekeeping genes, were determined from the Northern/semi-quantitative RT-PCR analyses. Sequence data obtained in this study are listed in the dbEST database (DV216698 to DV216767). As a single-cell C₄ system that lacks Kranz anatomy, a better understanding of how H. verticillata operates may facilitate the design of a transgenic C₄ system in a C₃ crop species.Srinath K. Rao and Hiroshi Fukayama contributed equally to this study.     

Immunocytochemical localization of enzymes involved in the C3 and C4 pathways in the photosynthetic cells of an amphibious sedge, Eleocharis vivipara

Eleocharis vivipara link, an amphibious leafless sedge, develops traits of C₄ photosynthesis and Kranz anatomy in the terrestrial form but develops C₃-like traits with non-Kranz anatomy when submerged. The cellular localization of C₃ and C₄ enzymes in the photosynthetic cells of the two forms was investigated by immunogold labeling and electron microscopy. The terrestrial form has mesophyll cells and three kinds of bundle sheath cell, namely, parenchyma sheath cells, non-chlorophyllous mestome sheath cells, and Kranz cells. Phosphoenol-pyruvate carboxylase (PEPCase) was present in the cytosol of both the mesophyll cells and the parenchyma sheath cells, with higher-density labeling in the latter, but not in the Kranz cells. Pyruvate, Pi dikinase (PPDK) was found at high levels in the chloroplasts of both the mesophyll cells and the parenchyma sheath cells with some-what stronger labeling in the latter. This enzyme was also absent from the Kranz cells. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was found in the chloroplasts of all types of photosynthetic cell, but labeling was significantly less intense in the parenchyma sheath cells than in other types of cell. The submerged form also has three types of photosynthetic cell, as well as non-chlorophyllous mestome sheath cells, but it lacks the traits of Kranz anatomy as a consequence of modification of the cells. Rubisco was densely distributed in the chloroplasts of all the photosynthetic cells. However, PEPCase and PPDK were found in both the mesophyll cells and the parenchyma sheath cells but at lower levels than in the terrestrial form. These data reveal that the terrestrial form has a unique pattern of cellular localization of C₃ and C₄ enzymes, and they suggest that this pattern and the changes in the extent of accumulation of the various enzymes are the main factors responsible for the difference in photosynthetic traits between the two forms.Abbreviations CAM crassulacean acid metabolism - MC meso phyll cell - PSC parenchyma sheath cell - KC Kranz cell - PEP-Case phosphoenolpyruvate carboxylase - PPDK pyruvate, Pi dikinase - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - LS large subunit - RuBP ribulose-1,5-bisphosphate This study was supported by Grants-in-Aid from the Ministry of Agriculture, Forestry and Fisheries of Japan (Integrated Research Program for the Use of Biotechnological Procedures for Plant Breeding) and from the Science and Technology Agency of Japan (Enhancement of Center-of-Excellence, the Special Coordination Funds for Promoting Science and Technology). The author is grateful to Drs M. Matsuoka and S. Muto for providing the antisera and Dr. M. Samejima for his advice at the early stages of this study.     

Hydrogen-isotope composition of leaf water in C3 and C4 plants: its relationship to the hydrogen-isotope composition of dry matter

The natural abundance hydrogen-isotope composition of leaf water ( ) and leaf organic matter ( _D ^org ) was measured in leaves of C₃ and C₄ dicotyledons and monocotyledons. The value of leaf water showed a marked diurnal variation, greatest enrichment being observed about midday. However, this variation was greater in the more slowly transpiring C₄ plants than in C₃ plants under comparable environmental conditions. A model based on analogies with a constant feed pan of evaporating water was developed and the difference between C₃ and C₄ plants expressed in terms of either differences in kinetic enrichment or different leaf morphology. Microclimatic and morphological features of the leaves which may be associated with this factor are discussed. There was no daily excursion in the _D ^org value in leaves of either C₃ or C₄ plants. When _D ^org values were referenced to the mean values during the period of active photosynthesis, the discrimination against deuterium during photosynthetic metabolism (D) was greater in C₃ plants (-117 to -121) than in C₄ plants (-86 to -109).These results show that the different water use strategies of C₃ and C₄ plants are responsible for the measured difference in deuterium-isotope composition of leaf water. However, it is unlikely that these physical processes account fully for the differences in hydrogen-isotope composition of the products of C₃ and C₄ photosynthetic metabolism.Symbols Hydrogen-isotope composition of leaf water - _D ^org hydrogen-isotope composition of leaf organic matter     

Light-dependent pH changes in leaves of C4 plants Comparison of the pH response to carbon dioxide and oxygen with that of C3 plants

Cytosolic and vacuolar pH changes caused by illumination or a changed composition of the gas phase were monitored in leaves of the NAD malic-enzyme-type C₄ plant Amaranthus caudatus L. and the C₃ plant Vicia faba L. by recording changes in the fluorescence of pH-indicating dyes which had been fed to the leaves. Light-dependent cytosolic alkalization and vacuolar acidification were maximal in the mesophyll cells under high-fluence-rate illumination and in the absence of CO₂. Under the same conditions, measurements of light scattering and electrochromic absorption changes at 518 nm revealed maximum thylakoid energization. The results show an intimate relationship between the energization of the photosynthetic apparatus by light, an increase in cytosolic pH and a decrease in vacuolar pH. This was true for both the C₄ and the C₃ plant, although kinetics, extent and even direction of cytosolic pH changes differed considerably in these plants, reflecting the differences in photosynthetic carbon metabolism. Darkening produced rapid acidification in Vicia, but not in Amaranthus. Continued alkalization in Amaranthus is interpreted to be the result of the decarboxylation of a C₄ intermediate and the release of liberated CO₂. In the presence of CO₂, energy consumption by carbon reduction decreased thylakoid energization, cytosolic alkalization and vacuolar acidification. Under low-fluence-rate illumination, thylakoid energization and light-dependent cytosolic and vacuolar pH changes were decreased in CO₂-free air compared with thylakoid energization and pH changes in 1% oxygen/99% nitrogen not only in the C₃ plant, but also in Amaranthus. Since oxygenation of ribulose bisphosphate initiates energy-consuming photorespiratory reactions in 21% oxygen, but not in 1% oxygen, this shows that photorespiratory reactions are active not only in the C₃ but also in the C₄ plant in the absence of external CO₂. Photorespiratory conditions appeared to decrease energization not only in the chloroplasts, but also in the cytosol. This is indicated by decreased transfer of protons from the cytosol into the vacuole, a process which is energy-dependent.Abbreviations CDCF 5-(and 6-)carboxy-2,7-dichlorofluorescein - P₇₀₀ electron-donor pigment in the reaction center of photosystem I - RuBP ribulose-1,5-bisphosphate This work was supported, within the framework of the Sonderforschungsbereiche 176 and 251 of the University of Würzburg, by the Gottfried-Wilhelm-Leibniz Program of the Deutsche Forschungsgemeinschaft. A.S.R. was the recipient of a fellowship from the Alexander-von-Humboldt-Foundation. We are grateful to Mr. Carsten Werner and Mrs. Spidola Neimanis for cooperation.     

Incorporation of 14CO2 into C4 acids by leaves of C3-C4 intermediate and C3 species of Moricandia and Panicum at the CO2 compensation concentration

Comparative ¹⁴CO₂ pulse-¹²CO₂ chase studies performed at CO₂ compensation ()-versus air-concentrations of CO₂ demonstrated a four-to eightfold increase in assimilation of ¹⁴CO₂ into the C₄ acids malate and aspartate by leaves of the C₃-C₄ intermediate species Panicum milioides Nees ex Trin., P. decipiens Nees ex Trin., Moricandia arvensis (L.) DC., and M. spinosa Pomel at . Specifically, the distribution of ¹⁴C in malate and aspartate following a 10-s pulse with ¹⁴CO₂ increases from 2% to 17% (P. milioides) and 4% to 16% (M. arvensis) when leaves are illuminated at the CO₂ compensation concentration (20 l CO₂/l, 21% O₂) versus air (340 l CO₂/l, 21% O₂). Chasing recently incorporated ¹⁴C for up to 5 min with ¹²CO₂ failed to show any substantial turnover of label in the C₄ acids or in carbon-4 of malate. The C₄-acid labeling patterns of leaves of the closely related C₃ species, P. laxum Sw. and M. moricandioides (Boiss.) Heywood, were found to be relatively unresponsive to changes in pCO₂ from air to . These data demonstrate that the C₃-C₄ intermediate species of Panicum and Moricandia possess an inherently greater capacity for CO₂ assimilation via phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) at the CO₂ compensation concentration than closely related C₃ species. However, even at , CO₂ fixation by PEP carboxylase is minor compared to that via ribulosebisphosphate carboxylase (EC 4.1.1.39) and the C₃ cycle, and it is, therefore, unlikely to contribute in a major way to the mechanism(s) facilitating reduced photorespiration in the C₃-C₄ intermediate species of Panicum and Moricandia.Abbreviations Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - PEP phosphoenolpyruvate - CO₂ compensation concentration - 3PGA 3-phosphoglycerate - SuP sugar monophosphates - SuP₂ sugar bisphosphates Published as Paper No. 8249, Journal Series, Nebraska Agricultural Research Division     

Coordination of the cell-specific distribution of the four subunits of glycine decarboxylase and of serine hydroxymethyltransferase in leaves of C3-C4 intermediate species from different genera

The cell-specific distribution of the four subunit proteins (P, L, T and H) of glycine decarboxylase (GDC) and of serine hydroxymethyltransferase (SHMT) has been studied in the leaves of C₃-C₄ intermediate and C₄ species of three genera (Flaveria, Moricandia and Panicum) using immunogold localization. Antibodies raised against these proteins from pea leaf mitochondria were used to probe Western blots of total leaf proteins of F. linearis Lag., M. arvensis (L.) DC and P. milioides Nees ex Trin. (C₃-C₄), and F. trinervia (Spring.) Mohr and P. miliaceum (L.) (C₄). For all species, each antibody recognised specifically a protein of similar molecular weight to that in pea leaves. In leaves of M. arvensis the P protein was present in the mitochondria of the bundle-sheath cells but was undetectable in those of the mesophyll, whereas the L, T and H proteins and SHMT were present in both cell types. The density of immunogold labelling of SHMT on the mitochondria of mesophyll cells was less than that on those of the bundle-sheath cells, which correlates with the relative activities of SHMT in these cell types. These data reveal that the lack of functional GDC in the mesophyll cells of M. arvensis, which is the principal biochemical reason for reduced photorespiration in this species, is due to the loss of a single subunit protein. This lack of coordinate expression of the subunit proteins of GDC within a photosynthetic cell represents a clear difference between M. arvensis and other C₃ and C₃-C₄ species. None of the GDC proteins was detectable in the mesophyll cells of the C₃-C₄ and C₄ Flaveria and Panicum species but all were present in the bundle-sheath cells. The differences in the distribution of the GDC proteins in leaves of the C₃-C₄ species studied are discussed in relation to the evolution of photosynthetic mechanisms.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

Modelling C3 photosynthesis: A sensitivity analysis of the photosynthetic carbon-reduction cycle

A model of the C ₃ photosynthetic system is developed which describes the sensitivity of the steadystate rate of carbon dioxide assimilation to changes in the activity of several enzymes of the system. The model requires measurements of the steady-state rate of carbon dioxide assimilation, the concentrations of several intermediates in the photosynthetic system, and the concentration of the active site of ribulose 1,5-bisphosphate carboxyalse/oxygenase (Rubisco). It is shown that in sunflowers (Helianthus annuus L.) at photon flux densities that are largely saturating for the rate of photosynthesis, the steady-stete rate of carbon dioxide assimilation is most sensitive to Rubisco activity and, to a lesser degree, to the activities of the stromal fructose, 6-bisphosphatase and the enzymes catalysing sucrose synthesis. The activities of sedoheptulose 1,7-bisphosphatase, ribulose 5-phosphate kinase, ATP synthase and the ADP-glucose pyrophosphorylase are calculated to have a negligible effect on the flux under the high-light conditions. The utility of this analysis in developing simpler models of photosynthesis is also discussed.Abbreviations c _i intercellular CO₂ concentration - C _infP ^supJ control coefficient for enzyme P with respect to flux J - DHAP dihydroxyacetonephosphate - E4P erythrose 4-phosphate - F6P fructose 6-phosphate - FBP fructose 1,6-bisphosphate - FBPase fructose 1,6-bisphosphatase - G3P glyceraldehyde 3-phosphate - G1P glucose 1-phosphate - G6P glucose 6-phosphate - Pi inorganic phosphate - PCR photosynthetic carbon reduction - PGA 3-phosphoglyceric acid - PPFD photosynthetically active photon flux density - R _n ^J response coefficient for effector n with respect to flux J - R5P ribose 5-phosphate - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - Ru5P ribulose 5-phosphate - RuBP ribulose 1,5-bisphosphate - S7P sedoheptulose 7-phosphate - SBP sedoheptulose 1,7-bisphosphate - SBPase sedoheptulose 1,7-bisphosphatase - SPS sucrose-phosphate synthase - Xu5P xylulose 5-phosphate - _n ^P elasticity coefficient for effector n with respect to the catalytic velocity of enzyme P This research was funded by an Australian Research Council grant to I.E.W. and was undertaken during a visity by K.A.M. to the James Cook University of North Queensland. The expert help of Glenys Hanley and Mick Kelly is greatly appreciated.     

Reactivity of glyoxylate with hydrogen perioxide and simulation of the glycolate pathway of C3 plants and Euglena

The nonenzymatic reaction of glyoxylate and H₂O₂ was measured under physiological conditions of the pH and concentrations of reactants. The reaction of glyoxylate and H₂O₂ was secondorder, with a rate constant of 2.27 l mol^-1 s^-1 at pH 8.0 and 25° C. The rate constant increased by 4.4 times in the presence of Zn²⁺ and doubled at 35°C. We propose a mechanism for the reaction between glyoxylate and H₂O₂. From a comparison of the rates of H₂O₂ decomposition by catalase and the reaction with glyoxylate, we conclude that H₂O₂ produced during glycolate oxidation in peroxisomes is decomposed by catalase but not by the reaction with glyoxylate, and that photorespiratory CO₂ originates from glycine, but not from glyoxylate, in C₃ plants. Simulation using the above rate constant and reported kinetic parameters leads to the same conclusion, and also makes it clear that alanine is a satisfactory amino donor in the conversion of glyoxylate to glycine. Some serine might be decomposed to give glycine and methylene-tetrahydrofolate; the latter is ultimately oxidized to CO₂. In the simulation of the glycolate pathway of Euglena, the rate constant was high enough to ensure the decarboxylation of glyoxylate by H₂O₂ to produce photorespiratory CO₂ during the glycolate metabolism of this organism.Abbreviations Chl chlorophyll - GGT glutamate: glyoxylate aminotransferase (EC 2.6.1.4) - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - SGT serine: glyoxylate aminotransferase (EC 2.6.1.45) This is the ninth in a series on the metabolism of glycolate in Euglena gracilis. The eighth is Yokota et al. (1982)     

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C₄ phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro ³²P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH₄Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca²⁺ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca²⁺ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C₄ MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C₄-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C₄ mesophyll cytosol.Abbreviations BS bundle-sheath - CAM Crassulacean acid metabolism - DHAP dihydroxyacetone phosphate - FPLC fast-protein liquid chromatography - Glc6P glucose 6-phosphate - I_0.5 50% inhibition constant - MC mesophyll cell(s) - MP me-sophyll protoplast(s) - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC protein-Ser/Thr kinase - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - Pyr pyruvate - Ser serine The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.     

Metabolism of phosphoenolpyruvate in the C4 cycle during photosynthesis in the phosphoenolpyruvate-carboxykinase C4 grass Spartina anglica Hubb.

The aim of this work was to investigate the fate of phosphoenolpyruvate (PEP) produced by decarboxylation of oxaloacetate during photosynthesis in the bundle sheaths of leaves of the PEP-carboxykinase C₄ grass Spartina anglica Hubb. Mesophyll protoplasts and bundle sheath cells were separated enzymically and used to investigate activities and distributions of putative enzymes of the C₄ cycle and the photosynthetic carbon metabolism of bundle sheath cells. The results indicate that neither conversion of PEP to pyruvate nor its conversion to 3-phosphoglycerate can account for all of the carbon flux through the C₄ cycle during photosynthesis. It is likely, therefore, either that PEP moves directly from bundle sheath to mesophyll or that more than one pathway of regeneration of PEP is involved in the C₄ cycle in this plant.Abbreviations Chl chlorophyll - PEP phosphoenolpyruvate - Pi phosphate - RuBP ribulose-1,5-bisphosphate     

Identification and characterization of a novel antimicrobial peptide from the venom of the ant Tetramorium bicarinatum

A novel antimicrobial peptide, named Bicarinalin, has been isolated from the venom of the ant Tetramorium bicarinatum. Its amino acid sequence has been determined by de novo sequencing using mass spectrometry and by Edman degradation. Bicarinalin contained 20 amino acid residues and was C-terminally amidated as the majority of antimicrobial peptides isolated to date from insect venoms. Interestingly, this peptide had a linear structure and exhibited no meaningful similarity with any known peptides. Antibacterial activities against Staphylococcus aureus and S. xylosus strains were evaluated using a synthetic replicate. Bicarinalin had a potent and broad antibacterial activity of the same magnitude as Melittin and other hymenopteran antimicrobial peptides such as Pilosulin or Defensin. Moreover, this antimicrobial peptide has a weak hemolytic activity compared to Melittin on erythrocytes, suggesting potential for development into an anti-infective agent for use against emerging antibiotic-resistant pathogens.     

Monocotyledonous C4 NADP+ -malate dehydrogenase is efficiently synthesized,targeted to chloroplasts and processed to an active form in transgenic plants of the C3 dicotyledon tobacco

Chloroplastic NADP⁺-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbonfixation pathway of some C₄ plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C₃ plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C₃-C₄ photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic proteinimport machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of l-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme.     

Modulating the activity of avian pancreatic lipases by an alkyl chain reacting with an accessible sulfhydryl group

Both turkey (TPL) and chicken (CPL) pancreatic lipases possess only one exposed sulfhydryl residue (Cystein114). After preincubation with the lipase, the sulfhydryl reagent C12 -TNB was found to be a powerful inhibitor of TPL whereas it had no effect on the CPL activity. Based on the 3D structure modelling and the molecular dynamics, the bulky dodecyl chain might hamper the lid movement of the TPL leading to the lipase inhibition upon reaction with C12 -TNB. Meanwhile, the predicted position of the C12 chain linked to Cystein114 of CPL could not block the lid opening mechanism which explains the absence of inhibition by C12 -TNB. Surprisingly, when added during the substrate hydrolysis, C12 -TNB activated the TPL but not the CPL that was slightly inhibited under these conditions. The 3D structure model generated for the open forms of C12 -TPL and C12 -CPL complexes showed that Cystein114 is still accessible and might react with C12 -TNB. Our models clearly explain the activation of TPL and the partial inhibition of CPL after the binding of the C12 chain to the enzyme.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Probing the steric barrier of nonionic surfactant vesicles with melittin

The role of the surface polymer brush of nonionic surfactant vesicles (NSV) in inhibiting interactions with small membrane-perturbing molecules was investigated using the bee venom peptide melittin as a probe. The interaction between melittin and NSV was compared with that of distearoylphosphatidylcholine (DSPC) vesicles and sterically stabilised liposomes (SSL) containing 5 mol% pegylated distearoylphosphatidylethanolamine (DSPE.E₄₄). The degree of melittin interaction with the various vesicles was determined by measuring peptide binding and folding, using intrinsic tryptophan fluorescence and circular dichroism respectively, in addition to monitoring the release of encapsulated carboxyfluorescein dye. NSV composed of 1,2-di-O-octadecyl-rac-glyceryl-3-(ω-dodecaethylene glycol) (2C₁₈E₁₂) showed a strong affinity for melittin, whilst exhibiting ~ 50% less bound peptide than SSL. 2C₁₈E₁₂:Chol vesicles showed reduced melittin interaction, in a manner consistent with Chol incorporation into DSPC vesicles. These results are discussed with respect to the effect of Chol on the in-plane order of 2C₁₈E₁₂ bilayers and consequent attenuation of hydrophobic interactions with the peptide. NSV formed from equimolar mixtures of polyoxyethylene-n-stearoyl ethers C₁₈E₂ and C₁₈E₂₀ showed a greater interaction with melittin than 2C₁₈E₁₂. However, replacing C₁₈E₂₀ with C₁₈E₁₀ was sufficient to achieve an attenuation of melittin interaction similar to that observed in 2C₁₈E₁₂:Chol vesicles. This indicates that the presence of surface polymer brush alone may confer resistance to melittin, provided hydrophobic interactions between the peptide and the vesicles can be minimised, through improved in-plane bilayer order.     

Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l_o-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φ_o, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φ_o was related to the assumed partition coefficient k_p of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k_p = 0 (probe completely excluded from liquid-ordered phase), Φ_o = 0.16 and with k_p = 0.2, Φ_o = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.