Developing Native Multispecies Sod: An Alternative Rehabilitation Method for Disturbed Lands

Natural and anthropogenic disturbance provides a mechanism for resetting the species assemblage within natural communities. Following anthropogenic disturbance particularly, there are generally more weedy species. Restoration of ecosystem structure and function in disturbed areas could aid in the control of these undesirable species. Attempts to re‐establish vegetation via direct sowing, hydroseeding, drill seeding, imprinting or plugging of either native or non‐native species can be effective, but can result in increased erosion and weed proliferation prior to desirable cover of the intended species. The use of native species for rehabilitation can be preferable because non‐native species are often less suited to the local environment and can pose a threat to the integrity of adjacent ecosystems. Native multispecies sod could become a useful rehabilitation tool, but research is needed to determine which species to use and the appropriate number of species to include. Mixtures of 4–7 native species were grown for 7 months, and clipped dry biomass, species percent abundance, and total ground cover were assessed over time in order to determine the suitability of 20 native species for sod production. Despite differences between mixtures at some harvests, all mixtures established and produced similar biomass by the end of the study. Final percent cover varied widely among species; 16 of the 20 species increased significantly over the course of production. Differences in total ground cover between mixtures were explained by the growth of the particular species rather than any synergistic effect of species diversity.     

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

A note on catches of basking sharks, Cetorhinus maximus (Gunnerus), off Norway and their relation to possible migration paths

The distribution and the times of the main catches of the basking shark, Cetorhinus, maximus (Gunnerus), in the commercial fishery off the coast of Norway during the seasons of 1971, 1972 and 1973 are given and analysed. No easily recognized regularities have been identified from season to season so that it is evident that there is no pattern of migration towards the Norwegian coast comparable to that of the bluefin tuna, Thunnus thynnus L., which once provided a substantial fishery in the same area.     

Immunological responses and viral modulatory effects of vaccination with recombinant modified vaccinia virus Ankara (rMVA) expressing structural and regulatory transgenes of simian immunodeficiency virus (SIVmac32H/J5M)

Background The immunogenicity and protective efficacy of recombinant modified vaccinia virus Ankara (rMVA) vectors expressing structural (gag/pol, env) and regulatory (tat, rev, nef) genes of SIVmac251/32H‐J5 (rMVA‐J5) were assessed. Methods Immunization with rMVA constructs (2.5 × 10⁷ IU) 32, 20 and 8 weeks pre‐challenge was compared with 32 and 20 weeks but with a final boost 8 weeks pre‐challenge with 2 × 10⁶ fixed‐inactivated HSC‐F4 cells infected with SIVmac32H. Controls received rMVA vectors expressing an irrelevant transgene or were naïve challenge controls. All received 10 MID₅₀ SIVmac32H/J5 intravenously. Results Vaccinates immunized with rMVA‐J5 exhibited significant, albeit transient, control of peak primary viraemia despite inconsistent and variable immune responses elicted by vaccination. Humoral and cellular responses to Env were most consistent, with lower responses to Nef, Rev and Tat. Increasing titres of anti‐vaccinia neutralizing antibodies reflected the number and dose of rMVA inoculations. Conclusions Improved combinations of viral vectors are required to elicit appropriate immune responses to control viral replication.     

Distinct modes of recognition of the lipoyl domain as substrate by the E1 and E3 components of the pyruvate dehydrogenase multienzyme complex

Two-dimensional (15)N-heteronuclear single-quantum coherence (HSQC) NMR studies with a di-domain (lipoyl domain+ linker+ peripheral subunit-binding domain) of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus allowed a molecular comparison of the need for lipoic acid to be covalently attached to the lipoyl domain in order to undergo reductive acetylation by the pyruvate decarboxylase (E1) component, in contrast with the ability of free lipoic acid to serve as substrate for the dihydrolipoyl dehydrogenase (E3) component. Tethering the lipoyl domain to the peripheral subunit-binding domain in a complex with E1 or E3 rendered the system more like the native enzyme complex, compared with the use of a free lipoyl domain, yet of a size still amenable to investigation by NMR spectroscopy. Recognition of the tethered lipoyl domain by E1 was found to be ensured by intensive interaction with the lipoyl-lysine-containing beta-turn and with residues in the protruding loop close to the beta-turn. The size and sequence of this loop varies significantly between species and dictates the lipoylated lipoyl domain as the true substrate for E1. In contrast, with E3 the main interaction sites on the tethered lipoyl domain were revealed as residues Asp41 and Ala43, which form a conserved sequence motif, DKA, around the lipoyl-lysine residue. No domain specificity is observed at this step and substrate channelling in the complex thus rests on the recognition of the lipoyl domain by the first enzyme, E1. The cofactor, thiamine diphosphate, and substrate, pyruvate, had distinct but contrasting effects on the E1/di-domain interaction, whereas NAD(+) and NADH had negligible effect on the E3/di-domain interaction. Tethering the lipoyl domain did not significantly change the nature of its interaction with E1 compared with a free lipoyl domain, indicative of the conformational freedom allowed by the linker in the movement of the lipoyl domain between active sites.     

Engineering the structural stability and functional properties of the GI domain into the intrinsically unfolded GII domain of the yeast linker histone Hho1p

Yeast Hho1p contains two domains, GI and GII, that are homologous to the single globular domain of the linker histone H1 (GH1). We showed previously that the isolated GI and GII domains have different structural stabilities and functional properties. GI, like GH1 and the related GH5, is stably folded at low ionic strength (10 mM sodium phosphate) and gives strong protection of chromatosome-length DNA ( approximately 166 bp) during micrococcal nuclease digestion of chromatin. GII is intrinsically unfolded in 10 mM sodium phosphate and gives weak chromatosome protection, but in 250 mM sodium phosphate has a structure very similar to that of GI as determined by NMR spectroscopy. We now show that the loop between helices II and III in GII is the cause of both its instability and its inability to confer strong chromatosome protection. A mutant GII, containing the loop of GI, termed GII-L, is stable in 10 mM sodium phosphate and is as effective as GI in chromatosome protection. Two GII mutants with selected mutations within the original loop were also slightly more stable than GII. In GII, two of the four basic residues conserved at the second DNA binding site ("site II") on the globular domain of canonical linker histones, and in GI, are absent. Introduction of the two "missing" site II basic residues into GII or GII-L destabilised the protein and led to decreased chromatosome protection relative to the protein without the basic residues. In general, the ability to confer chromatosome protection in vitro is closely related to structural stability (the relative population of structured and unstructured states). We have determined the structure of GII-L by NMR spectroscopy. GII-L is very similar to GII folded in 250 mM sodium phosphate, with the exception of the substituted loop region, which, as in GI, contains a single helical turn.     

Solution structure of the isolated Pelle death domain

The interaction between the death domains (DDs) of Tube and the protein kinase Pelle is an important component of the Toll pathway. Published crystallographic data suggests that the Pelle-Tube DD interface is plastic and implies that in addition to the two predominant Pelle-Tube interfaces, a third interaction is possible. We present the NMR solution structure of the isolated death domain of Pelle and a study of the interaction between the DDs of Pelle and Tube. Our data suggests the solution structure of the isolated Pelle DD is similar to that of Pelle DD in complex with Tube. Additionally, they suggest that the plasticity observed in the crystal structure may not be relevant in the functioning death domain complex.     

Exploring sequence/folding space: folding studies on multiple hydrophobic core mutants of ubiquitin

The stability, dynamic, and structural properties of ubiquitin and two multiple hydrophobic core mutants were studied. One of the mutants (U4) has seven substitutions in the hydrophobic core (M1L, I3L, V5I, I13F, L15V, V17M, and V26L). On average, its side chains are larger than the wild-type, and it can thus be thought of as having an overpacked core. The other mutant (U7) has two substitutions (I3V and I13V). On average, it has smaller side chains than the wild-type, and it can therefore be considered to be underpacked. The three proteins are well-folded and show similar backbone dynamics (T(1), T(2), and HNOE values), indicating that the regular secondary structure extends over the same residue ranges. The crystallographic structure of U4 was determined. The final R(factor) and R(free) are 0.198 and 0.248, respectively, at 2.18 A resolution. The structure of U4 is very similar to wild-type ubiquitin. Remarkably, there are almost no changes in the positions of the C(alpha) atoms along the entire backbone, and the hydrogen-bonding network is maintained. The mutations of the hydrophobic core are accommodated by small movements of side chains in the core of mutated and nonmutated residues. Unfolding and refolding kinetic studies revealed that U4 unfolds with the highest rates; however, its refolding rate constants are very similar to those of the wild-type protein. Conversely, U7 seems to be the most destabilized protein; its refolding rate constant is smaller than the other two proteins. This was confirmed by stopped-flow techniques and by H/D exchange methodologies. This work illustrates the possibility of repacking the hydrophobic core of small proteins and has important implications in the de novo design of stable proteins.