Affinity labelling of rat-muscle hexokinase type II by a glucose-derived alkylating agent.

The glucose-derived alkylating agent N-bromoacetylglucosamine (GlcNBrAc) is shown to cause a time-dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme-inhibitor complex prior to modification, indicating that the reagent is active-site-directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N-propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme-glucose complex from these studies is in agreement with the kinetically-determined Km of 0.2 mM. The substrate-unrelated but chemically similar alkylating agents bromoacetic acid and N-bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7--9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo-first-order rate constant) of 1.5 x 10(-3) S-1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active-site-directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I.     

Assignment of resonances in the 31P NMR spectrum of d(GGAATTCC) by regiospecific labeling with oxygen-17

The chemical synthesis of the octanucleotide d(GGAATTCC) in which each of the phosphate groups is sequentially replaced by an 17O-containing phosphate group using a polymer-supported phosphoramidite method is described. All seven phosphorus resonances in the 31P spectrum of d(GGAATTCC) can be resolved. Assignment of these resonances to a particular phosphate group in the chain is possible because labeling of a phosphate with 17O causes its particular signal to disappear from the spectrum. Phosphate residues toward the middle of the octamer have 31P NMR shifts similar to those found in polydeoxynucleotides, whereas those toward the ends resemble those of dinucleoside phosphates. These data are interpreted in terms of less flexibility of the phosphate groups in the center of the molecule as compared to those at the ends.     

Oriciopsin,a new ring-D cleaved tetranortriterpenoid,and flindersiamine from Oriciopsis glaberrima

A novel ring-D cleaved tetranortriterpenoid, oriciopsin and flindersiamine have been isolated from the whole plant extracts of Oriciopsis glaberrima. The structure of the new limonoid was determined from its ¹H NMR and ¹³C NMR spectra and by mass spectrometry.     

Neighbour specificity between Lolium perenne and Trifolium repens from a natural pasture

Summary Five genotypes of Trifolium repens and Lolium perenne were collected as neighbouring pairs along a fertility gradient in a natural pasture. After vegetative multiplication, the 25 possible combinations of Lolium genotype x Trifolium genotype were planted in the greenhouse in order to investigate competition between the genotypes. The comparison of the five combinations whose individual components had been neighbours with the combinations of genotypes that had not coexisted before disclosed no difference in total biomass production over 7 months. However, the yield of Trifolium increased when grown with the Lolium genotype which had been its natural neighbour, while the latter showed a decrease in yield. This neighbour specificity existed even when carryover effects from the sampling site had been eliminated (preconditioning period of 18 months) and when native Rhizobium strains were not present (inoculation with a non-native strain). The complex pattern of neighbour specificity with time indicated the importance of environmental conditions for its outcome. These results are a further confirmation of positive effects on the growth of Trifolium repens when grown together with its natural neighbour. They are discussed in the context of coexistence and coevolution     

Opportunistic predation by small fishes on epibiota of jetty pilings in urban waterways

Epibiota were sampled on nine small jetties in the tidal, urban canals of south-east Queensland, Australia, to determine if the small fishes that are associated with these jetties prey on the epibiota on the pilings of the jetties and whether these fishes depend on the epibiota as a source of food. Epibiota was dominated by barnacles, filamentous and foliose algae and ranged in thickness from 4 to 11 mm. The two species of fishes that associated most closely with jetty pilings, Pandaka lidwilli (Gobiidae) and Monodactylus argenteus (Monodactylidae), were sampled twice during the day and twice during the night for analysis of stomach contents. During the day, the diet of P. lidwilli was dominated by amphipods ( c. 70%, by mass of organic content), with copepods, bivalves and bryozoans each contributing <10%. At night, amphipods contributed less ( c. 45%) and copepods more ( c. 35%). The diet of M. argenteus was dominated by filamentous algae (55%) and amphipods (20%) during the day and filamentous algae (70%) and barnacle cirri (23%) at night. Epibiota, therefore, made a substantial contribution to the diet of the fishes but were not the sole source of food for either species. As jetties were the only structures that supported epibiota in the area, fishes probably sourced their epibiota from the pilings of the jetties. Whether fishes depended on the epibiota was, therefore, tested using a manipulative before-after-control-impact (BACI) study. Three jetties were assigned randomly to each of three treatments: (1) epibiota removed from pilings, (2) epibiota cut and damaged (a procedural control) and (3) epibiota left undisturbed. Abundances of P. lidwilli and M. argenteus around jetty pilings remained similar across all treatments from before to after the removal of epibiota. These results indicate that although fishes consumed epibiota on the jetties, they did not depend on the epibiota of the jetties for food.     

Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells

We have used the microtubule-stabilizing drug taxol to examine the relationship between microtubules and the appearance and cell surface distribution of acetylcholine receptors (AChRs) in primary cultures of chick embryonic muscle cells. Taxol at a 5-microM concentration induced the large scale polymerization of tubulin in muscle cells that was most obvious as intermittent bundles of microtubules along the myotube. Prominent bundles of microtubules were also clearly visible in the fibroblasts. This concentration of taxol had no significant effect on the incorporation rate, increased synthesis induced by brain extract or the total cell surface number of AChRs measured over a 24-h period. Thus, excess polymerization of microtubules does not affect the movement of receptors to the cell surface. However, when cell surface AChR distribution was examined using rhodamine-conjugated alpha-bungarotoxin, taxol treatment of myotubes was shown to induce the aggregation of receptors. If receptors were labeled before taxol addition, aggregation of these prelabeled receptors was also seen, a result indicating that taxol can induce the movement of receptors already in the membrane. We believe this evidence further implicates microtubules as being involved in the movement of these cell surface receptors in the plane of the myotube membrane.     

Bacteria and heterotrophic microflagellate production in the Santa Rosa Sound, Florida

Bacterial and microflagellate biomass and production and grazing onbacteria were compared weekly at a fixed station in Santa Rosa Sound,Florida, starting in February and ending in October. For bothpopulations the weekly variation in biomass and production was aslarge as the seasonal variation. Cycles for biomass and production ofthese organisms were generally out of phase, rendering it difficultto estimate the net grazing of bacteria by microflagellates atindividual time points. For evaluation of factors that control thefate of carbon cycled by bacterial, experiments were conducted toexamine bacterial growth rates in the absence of predators. Thisexamination resulted in low bacterial growth rates when biomass washigh, and rapid growth rates typically occurred near minimumpopulations. Further analysis suggested that microflagellatepredation was greater than bacterial production during minimumbacterial growth rates. With integration of production and grazingrates over the study period, factors controlling bacterial growthwere examined. Using this approach, 71% of the bacterial productionwas grazed by < 8.0µm predators. The microflagellate biomassproduction was 41% of the grazing rate on bacteria. The total amountof bacterial production assimilated into microflagellate biomass was29%. However, based on the variations in biomass and activity of themicrobial assemblages, it appears that substrate and predation exertalternating control on bacterial abundance and production.