Effects of polymer molecular weight on the size, activity, and stability of PEG-functionalized trypsin

Polymer conjugation increases an enzyme's circulation time and stability for use as a therapeutic agent, but this attachment indubitably affects its properties. Covalent attachment of multiple polyethylene glycol chains with sizes of either 2, 5, 10, or 20 kDa increases the molecular weight and hydrodynamic radius of the model enzyme trypsin. The sizes of these polymer-enzyme conjugates are increased to be within the recommended limits for PDEPT applications. The T(d) increases from 49 to 60 °C to expand the enzyme's workable range of conditions. This functionalization with PEG polymers of varying lengths maintains trypsin's enzymatic activity. Conjugate activities are 79-120% that of native trypsin at room temperature and 221-432% that of trypsin at 37 °C.     

The cytology and cytochemistry of the hemocytes of Mytilus edulis and their responses to experimentally injected carbon particles

Hemocytes of Mytilus edulis were examined cytologically and cytochemically. On the basis of structure, staining reactions, and phagocytic behavior, they were divided into two main groups: basophilic hemocytes and eosinophilic granular hemocytes (granulocytes). The basophilic cells were further divided into small lymphocytes and larger phagocytic macrophages reactive for lysosomal hydrolases. Mitosis was observed in granulocytes and in small lymphoid cells, believed to be the stem cells for the basophilic cell line. A few cells appeared to be intermediate between lymphocytes and small granulocytes. Macrophages were the main cell type involved in the clearance of injected carbon particles. However, granulocytes did show some phagocytic activity. Brown cells displaying apparent amoebocytic behavior were found to contain Fe³⁺ and Pb²⁺ in cytoplasmic inclusions, some of which were also reactive for β-glucuronidase and glucosaminidase. These cells appear to have a separate origin from the hemocytes.     

Conformations generated during turnover of the Azotobacter vinelandii nitrogenase MoFe protein and their relationship to physiological function

Various S=3/2 EPR signals elicited from wild-type and variant Azotobacter vinelandii nitrogenase MoFe proteins appear to reflect different conformations assumed by the FeMo-cofactor with different protonation states. To determine whether these presumed changes in protonation and conformation reflect catalytic capacity, the responses (particularly to changes in electron flux) of the alphaH195Q, alphaH195N, and alphaQ191K variant MoFe proteins (where His at position 195 in the alpha subunit is replaced by Gln/Asn or Gln at position alpha-191 by Lys), which have strikingly different substrate-reduction properties, were studied by stopped-flow or rapid-freeze techniques. Rapid-freeze EPR at low electron flux (at 3-fold molar excess of wild-type Fe protein) elicited two transient FeMo-cofactor-based EPR signals within 1 s of initiating turnover under N(2) with the alphaH195Q and alphaH195N variants, but not with the alphaQ191K variant. No EPR signals attributable to P cluster oxidation were observed for any of the variants under these conditions. Furthermore, during turnover at low electron flux with the wild-type, alphaH195Q or alphaH195N MoFe protein, the longer-time 430-nm absorbance increase, which likely reflects P cluster oxidation, was also not observed (by stopped-flow spectrophotometry); it did, however, occur for all three MoFe proteins under higher electron flux. No 430-nm absorbance increase occurred with the alphaQ191K variant, not even at higher electron flux. This putative lack of involvement of the P cluster in electron transfer at low electron flux was confirmed by rapid-freeze (57)Fe M?ssbauer spectroscopy, which clearly showed FeMo-factor reduction without P cluster oxidation. Because the wild-type, alphaH195Q and alphaH195N MoFe proteins can bind N(2), but alphaQ195K cannot, these results suggest that P cluster oxidation occurs only under high electron flux as required for N(2) reduction.     

In vitro anthelmintic effects of cysteine proteinases from plants against intestinal helminths of rodents

Infections with gastrointestinal (GI) nematodes are amongst the most prevalent worldwide, especially in tropical climates. Control of these infections is primarily through treatment with anthelmintic drugs, but the rapid development of resistance to all the currently available classes of anthelmintic means that alternative treatments are urgently required. Cysteine proteinases from plants such as papaya, pineapple and fig are known to be substantially effective against three rodent GI nematodes, Heligmosomoides polygyrus, Trichuris muris and Protospirura muricola, both in vitro and in vivo. Here, based on in vitro motility assays and scanning electron microscopy, we extend these earlier reports, demonstrating the potency of this anthelmintic effect of plant cysteine proteinases against two GI helminths from different taxonomic groups - the canine hookworm, Ancylostoma ceylanicum, and the rodent cestode, Rodentolepis microstoma. In the case of hookworms, a mechanism of action targeting the surface layers of the cuticle indistinguishable from that reported earlier appears to be involved, and in the case of cestodes, the surface of the tegumental layers was also the principal location of damage. Hence, plant cysteine proteinases have a broad spectrum of activity against intestinal helminths (both nematodes and cestodes), a quality that reinforces their suitability for development as a much-needed novel treatment against GI helminths of humans and livestock.     

Purification and characterization of the FeII- and alpha-ketoglutarate-dependent xanthine hydroxylase from Aspergillus nidulans

His6-tagged xanthine/alpha-ketoglutarate (alphaKG) dioxygenase (XanA) of Aspergillus nidulans was purified from both the fungal mycelium and recombinant Escherichia coli cells, and the properties of the two forms of the protein were compared. Evidence was obtained for both N- and O-linked glycosylation on the fungus-derived XanA, which aggregates into an apparent dodecamer, while bacterium-derived XanA is free of glycosylation and behaves as a monomer. Immunological methods identify phosphothreonine in both forms of XanA, with phosphoserine also detected in the bacterium-derived protein. Mass spectrometric analysis confirms glycosylation and phosphorylation of the fungus-derived sample, which also undergoes extensive truncation at its amino terminus. Despite the major differences in the properties of these proteins, their kinetic parameters are similar (kcat = 30-70 s-1, Km of alphaKG = 31-50 muM, Km of xanthine approximately 45 muM, and pH optima at 7.0-7.4). The enzyme exhibits no significant isotope effect when [8-2H]xanthine is used; however, it demonstrates a 2-fold solvent deuterium isotope effect. CuII and ZnII potently inhibit the FeII-specific enzyme, whereas CoII, MnII, and NiII are weaker inhibitors. NaCl decreases the kcat and increases the Km of both alphaKG and xanthine. The alphaKG cosubstrate can be substituted with alpha-ketoadipate (9-fold decrease in kcat and 5-fold increase in the Km compared to those of the normal alpha-keto acid), while the alphaKG analogue N-oxalylglycine is a competitive inhibitor (Ki = 0.12 muM). No alternative purines effectively substitute for xanthine as a substrate, and only one purine analogue (6,8-dihydroxypurine) results in significant inhibition. Quenching of the endogenous fluorescence of the two enzyme forms by xanthine, alphaKG, and DHP was used to characterize their binding properties. A XanA homology model was generated on the basis of the structure of the related enzyme TauD (PDB entry 1OS7) and provided insights into the sites of posttranslational modification and substrate binding. These studies represent the first biochemical characterization of purified xanthine/alphaKG dioxygenase.     

Testing the role of genetic factors across multiple independent invasions of the shrub Scotch broom (Cytisus scoparius)

Knowledge of the introduction history of invasive plants informs on theories of invasiveness and assists in the invasives management. For the highly successful invasive shrub Scotch broom, Cytisus scoparius, we analysed a combination of nuclear and chloroplast microsatellites for eight native source regions and eight independent invasion events in four countries across three continents. We found that two exotic Australian populations came from different sources, one of which was derived from multiple native populations, as was an invasive sample from California. An invasive population from New Zealand appeared to be predominantly sourced from a single population, either from the native or exotic ranges. Four invasive populations from Chile were genetically differentiated from the native range samples analysed here and so their source of introduction could not be confirmed, but high levels of differentiation between the Chilean populations suggested a combination of different sources. This extensive global data set of replicated introductions also enabled tests of key theories of invasiveness in relation to genetic diversity. We conclude that invasive populations have similar levels of high genetic diversity to native ranges; levels of admixture may vary across invasive populations so admixture does not appear to have been an essential requirement for invasion; invasive and native populations exhibit similar level of genetic structure indicating similar gene flow dynamics for both types of populations. High levels of diversity and multiple source populations for invasive populations observed here discount founder effects or drift as likely explanations for previously observed seed size differences between ranges. The high levels of genetic diversity, differential and source admixture identified for most exotic populations are likely to limit the ability to source biocontrol agents from the native region of origin of invasive populations.     

Odour emissions from anaerobic piggery ponds. 1. Results of a three season, 14-month survey

Odour emission rates were measured for seven different anaerobic ponds treating piggery wastes at six to nine discrete locations across the surface of each pond on each sampling occasion over a 14-month period. Emission rate values varied between ponds, between seasons for the same pond and even for the same pond on different days of a sampling week. Average seasonal emission rates ranged from 7.9 to 46.5OU/m(2)s, while average emission rates ranged from 16.0 to 29.0OU/m(2)s. Factors potentially responsible for the variability in emission rates were investigated, including air and pond liquor temperatures, time of day of sample collection, season and the impact of a prolonged drought.     

Drosophila melanogaster, a model system for comparative studies on the responses to real and simulated microgravity

A key requirement to enhance our understanding of the response of biological organisms to different levels of gravity is the availability of experimental systems that can simulate microgravity and hypergravity in ground-based laboratories. This paper compares the results obtained from analysing gene expression profiles of Drosophila in space versus those obtained in a random position machine (RPM) and by centrifugation. The correlation found validates the use of the RPM simulation technique to establish the effects of real microgravity on biological systems. This work is being extended to investigate Drosophila development in another gravity modifying instrument, the levitation magnet.     

Contrasting fire-related resilience of ecologically dominant ants in tropical savannas of northern Australia

This paper examines the role of fire in mediating the relative abundance of two of the world's major ecologically dominant ant genera, Iridomyrmex and Oecophylla, where they coexist across the tropical savanna landscapes of northern Australia. These taxa have contrasting biogeographical histories, which are predicted to lead to contrasting responses to fire. Iridomyrmex is an autochthonous Australian genus that has radiated primarily in the arid zone; as such, its abundance is predicted to be promoted by frequent fire because this maintains an open habitat. In contrast, Oecophylla is a genus of leaf‐nesting ants occurring in the canopies of Old World tropical rainforest, and is a recent arrival to Australia in geological time; the abundance of these ants is predicted to decline under frequent fire. We test these predictions using results from a landscape‐scale fire experiment, where three experimental fire regimes (including no fire) were applied to replicated subcatchments over a 5‐year period. Using sweep nets, ants were sampled in the grass layer (the habitat layer of greatest overlap between Iridomyrmex and Oecophylla) in eucalypt woodland (canopy cover < 30%) and open eucalypt forest (canopy cover about 50%) habitats. A total of 27 species from 11 genera were collected during the study; eight were common enough for statistical analysis, and the abundances of four of these were significantly affected by fire treatment. As predicted, the abundance of Iridomyrmex was promoted by fire, whereas that of Oecophylla declined. These changes occurred only under late‐season (relatively high intensity) fires, and for Oecophylla occurred only in open forest (not woodland) habitat. This fire‐mediated relationship between Iridomyrmex and Oecophylla mirrors the much broader, ecosystem‐wide dynamic between eucalypt‐dominated savanna and rainforest in tropical Australia, with savannas dominated by fire‐resistant sclerophyll elements of Australian origin, and rainforest dominated by fire‐sensitive mesophyll elements of South‐East Asian origin.