Boron bridging of rhamnogalacturonan‐II,monitored by gel electrophoresis,occurs during polysaccharide synthesis and secretion but not post‐secretion

The cell‐wall pectic domain rhamnogalacturonan‐II (RG‐II) is cross‐linked via borate diester bridges, which influence the expansion, thickness and porosity of the wall. Previously, little was known about the mechanism or subcellular site of this cross‐linking. Using polyacrylamide gel electrophoresis (PAGE) to separate monomeric from dimeric (boron‐bridged) RG‐II, we confirmed that Pb²⁺ promotes H₃BO₃‐dependent dimerisation in vitro. H₃BO₃ concentrations as high as 50 mm did not prevent cross‐linking. For in‐vivo experiments, we successfully cultured ‘Paul's Scarlet’ rose (Rosa sp.) cells in boron‐free medium: their wall‐bound pectin contained monomeric RG‐II domains but no detectable dimers. Thus pectins containing RG‐II domains can be held in the wall other than via boron bridges. Re‐addition of H₃BO₃ to 3.3 μm triggered a gradual appearance of RG‐II dimer over 24 h but without detectable loss of existing monomers, suggesting that only newly synthesised RG‐II was amenable to boron bridging. In agreement with this, Rosa cultures whose polysaccharide biosynthetic machinery had been compromised (by carbon starvation, respiratory inhibitors, anaerobiosis, freezing or boiling) lost the ability to generate RG‐II dimers. We conclude that RG‐II normally becomes boron‐bridged during synthesis or secretion but not post‐secretion. Supporting this conclusion, exogenous [³H]RG‐II was neither dimerised in the medium nor cross‐linked to existing wall‐associated RG‐II domains when added to Rosa cultures. In conclusion, in cultured Rosa cells RG‐II domains have a brief window of opportunity for boron‐bridging intraprotoplasmically or during secretion, but secretion into the apoplast is a point of no return beyond which additional boron‐bridging does not readily occur.     

Resistance in sorghum to shoot fly Atherigona soccata: Evidence for the source of leaf surface wetness

Leaf surface wetness (LSW) of the central whorl leaf of sorghum seedlings has been associated with susceptibility to shoot fly. Previous physical and physiological evidence suggested that LSW originates from the plant. This was confirmed by radioactive labelling methods using tritium and carbon-14. Tritiated water applied to the soil of potted seedlings was translocated to the surface of the whorl leaf. There were significant differences in the amount of tritiated water collected from susceptible (CSH 5) and resistant (IS 18551) genotypes. Studies with carbon-14 labelling of sorghum seedlings indicated the presence of (small amounts of) solutes in the surface water that may affect larval movement and survival.     

Diel zooplankton community feeding activity and filtration rates of Pseudoboeckella volucris and Daphniopsis studeri on sub-antarctic Marion Island

Experiments measuring zooplankton filter-feeding rates using a ¹⁴C-labelled green alga were carred out in situ for the first time in three shallow sub-antarctic lakes. The number and biomass of zooplankton were high in samples from two lakes enriched by penguin and seal faeces, and low in samples from a peat-lined lava-lakelet not biotically enriched. A diel survey of the lava-lakelet over 48 h showed that no vertical migration or diel variation in filter-feeding rate occured in the strongly pigmented copepod Pseudoboeckella volucris Kiefer population, the most abundant entomostracan present. The unpigmented cladoceran Daphniopsis studeri Rühe had a high in situ filter-feeding rate, exhibited very marked vertical migration, and was primarily a nocturnal grazer.     

Efficient CO2 fixation by surface Prochlorococcus in the Atlantic Ocean

Nearly half of the Earth''s surface is covered by the ocean populated by the most abundant photosynthetic organisms on the planet—Prochlorococcus cyanobacteria. However, in the oligotrophic open ocean, the majority of their cells in the top half of the photic layer have levels of photosynthetic pigmentation barely detectable by flow cytometry, suggesting low efficiency of CO₂ fixation compared with other phytoplankton living in the same waters. To test the latter assumption, CO₂ fixation rates of flow cytometrically sorted ¹⁴C-labelled phytoplankton cells were directly compared in surface waters of the open Atlantic Ocean (30°S to 30°N). CO₂ fixation rates of Prochlorococcus are at least 1.5–2.0 times higher than CO₂ fixation rates of the smallest plastidic protists and Synechococcus cyanobacteria when normalised to photosynthetic pigmentation assessed using cellular red autofluorescence. Therefore, our data indicate that in oligotrophic oceanic surface waters, pigment minimisation allows Prochlorococcus cells to harvest plentiful sunlight more effectively than other phytoplankton.     

Boron bridging of rhamnogalacturonan-II in Rosa and arabidopsis cell cultures occurs mainly in the endo-membrane system and continues at a reduced rate after secretion

Background and aimsRhamnogalacturonan-II (RG-II) is a domain of primary cell-wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occurs mainly within the Golgi system, concurrently with secretion or within the cell wall. We therefore explored the kinetics of RG-II bridging.MethodsCell-suspension cultures of Rosa and arabidopsis were pulse-radiolabelled with [¹⁴C]glucose, then the boron bridging status of newly synthesized [¹⁴C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests.Key resultsOptimal culture ages for ¹⁴C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [¹⁴C]polysaccharide production occurred for the first ~90 min; thereafter the radiolabelled molecules were tracked as they ‘aged’ in the wall. Monomeric and (boron-bridged) dimeric [¹⁴C]RG-II domains appeared simultaneously, both being detectable within 4 min of [¹⁴C]glucose feeding, i.e. well before the secretion of newly synthesized [¹⁴C]polysaccharides into the apoplast at ~15–20 min. The [¹⁴C]dimer : [¹⁴C]monomer ratio of RG-II remained approximately constant from 4 to 120 min, indicating that boron bridging was occurring within the Golgi system during polysaccharide biosynthesis. However, [¹⁴C]dimers increased slightly over the following 15 h, indicating that limited boron bridging was continuing after secretion.ConclusionsThe results show where in the cell (and thus when in the ‘career’ of an RG-II domain) boron bridging occurs, helping to define the possible biological roles of RG-II dimerization and the probable localization of boron-donating glycoproteins or glycolipids.     

Synthesis, 18F‐Radiolabelling and Biological Evaluations of C‐6 Alkylated Pyrimidine Nucleoside Analogues

Synthesis of pyrimidine derivatives with a side‐chain attached to the C‐6 of pyrimidine ring (6–14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV‐1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 µM) exhibited better binding affinity for HSV‐1 TK than acyclovir (ACV, 170 µM) and ganciclovir (GCV, 48 µM). Catalytic turnover constant (k _cat) of 12 (0.08 s^? 1) was close to the k _cat values of ACV (0.10 s^? 1) and GCV (0.10 s^? 1). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV‐1 TK‐transduced and non‐transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV‐1 TK could be developed as non‐toxic PET‐tracer molecule. Thus, ¹⁸F labelling of the precursor 14 was performed by nucleophilic substitution using [¹⁸F] tetrabutylammonium fluoride as the fluorinating reagent.     

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies

Summary. The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer’s disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Radiolabelling of both nLDL and oxLDL using [¹⁸F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [¹⁸F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [¹⁸F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [¹⁸F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.     

Energy partitioning in the Antarctic collembolan Cryptopygus antarcticus

Abstract. 1. Consumption, production and assimilation rates were determined for two age groups of Crypropygus antarcticus to give an estimate of energy utilization, and to investigate low temperature adaptation in its energy partitioning.
2. Feeding selectivity shown in laboratory preference tests was supported by gut analysis of field animals from contrasting sites. Although moulting rate was not significantly affected by food type, rates of growth were slowest and mortality highest when fed on a non-preferred substrate.
3. Both a radio labelling and a more direct method for measuring dry weight consumed gave similar results for Cvpropygus feeding on algae. The consuniption rate for animals when feeding on algae was lower than that on moss peat. The assimilation efficiency for immature animals feeding on algae was 46% and for mature animals was 19%; the values when feeding on moss peat were 7% and lo%, respectively, The net production efficiency ranged from 35%(inimatures) to 13% (matures) and was similar on both substrates.
4. Food consumption exceeded assimilation over the range 2.5–10°C, but the two converged from 2.5 to 0°C. Immature Cryptopygus maintained a net positive energy balance over 0–10°C, whilst below 1S°C respiration exceeded assimilation for mature individuals.
5. An estimate of the annual dry matter consumption (7 g m^-1 y^-1) by Ctypropygus in a moss turf at Signy Island agrees with one based on respiration data alone (Davis, 1981). The consumption at an alga-dominated site was c . 26 g m^-2 y^-l, and Crypropygus may have a locally limiting effect on net priniary production at such sites.     

Preparation of radiolabelled very-low-density lipoprotein in high yield by extended rat liver perfusion

A method is described using an extended (8 h), rat liver perfusion that produces approx. 100 mg of selectively radiolabelled, mature, very-low-density lipoprotein suitable for use in subsequent whole organ perfusion experiments.