Amino-terminal nucleotide-binding sequences of a Lactobacillus deoxynucleoside kinase complex isolated by novel affinity chromatography

A highly efficient new affinity medium for deoxycytidine kinase, deoxycytidine 5'-tetraphosphate-Sepharose (dCp4-Sepharose), has been constructed. A dCp4-Sepharose column effects a one-step, 19,000-fold, purification to homogeneity of dCyd kinase from the ammonium sulfate fraction of Lactobacillus acidophilus R-26 extract, with 60% recovery. dCTP, a potent end-product inhibitor, is used as an eluent, and it also stabilizes the extremely labile purified enzyme. A noncompeting deoxyadenosine kinase activity accompanies the deoxycytidine kinase activity eluted. Native polyacrylamide gel electrophoresis shows a single protein band, which coincides with both deoxycytidine kinase and deoxyadenosine kinase activities at several gel concentrations. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide band of 26,000 daltons. Since the native enzyme is known to have an Mr of 50,000, it appears that the enzyme is composed of two subunits of similar size. Sequence analysis of the intact protein from the N-terminus reveals but a single amino acid species per residue up to the 17th residue; at the 18th, 21st, 26th, and 27th residue positions of the sequence, however, there appear to be two different amino acids in almost equal amounts. This may indicate that the enzyme is composed of two nonidentical subunits having the same amino acid sequence near the N-terminus. Residues 6-13 contain the highly conserved Gly-X-X-Gly-X-Gly-Lys sequence found at the active sites of kinases and other nucleotide-binding proteins.     

Homologous and Heterologous Protection of Nonhuman Primates by Ebola and Sudan Virus-Like Particles

Filoviruses cause hemorrhagic fever resulting in significant morbidity and mortality in humans. Several vaccine platforms that include multiple virus-vectored approaches and virus-like particles (VLPs) have shown efficacy in nonhuman primates. Previous studies have shown protection of cynomolgus macaques against homologous infection for Ebola virus (EBOV) and Marburg virus (MARV) following a three-dose vaccine regimen of EBOV or MARV VLPs, as well as heterologous protection against Ravn Virus (RAVV) following vaccination with MARV VLPs. The objectives of the current studies were to determine the minimum number of vaccine doses required for protection (using EBOV as the test system) and then demonstrate protection against Sudan virus (SUDV) and Taï Forest virus (TAFV). Using the EBOV nonhuman primate model, we show that one or two doses of VLP vaccine can confer protection from lethal infection. VLPs containing the SUDV glycoprotein, nucleoprotein and VP40 matrix protein provide complete protection against lethal SUDV infection in macaques. Finally, we demonstrate protective efficacy mediated by EBOV, but not SUDV, VLPs against TAFV; this is the first demonstration of complete cross-filovirus protection using a single component heterologous vaccine within the Ebolavirus genus. Along with our previous results, this observation provides strong evidence that it will be possible to develop and administer a broad-spectrum VLP-based vaccine that will protect against multiple filoviruses by combining only three EBOV, SUDV and MARV components.     

Trait similarity, shared ancestry and the structure of neighbourhood interactions in a subtropical wet forest: implications for community assembly

Ecology Letters (2010) 13: 1503-1514 ABSTRACT: The phylogenetic structure and distribution of functional traits in a community can provide insights into community assembly processes. However, these insights are sensitive to the spatial scale of analysis. Here, we use spatially explicit, neighbourhood models of tree growth and survival for 19 tree species, a highly resolved molecular phylogeny and information on eight functional traits to quantify the relative efficacy of functional similarity and shared ancestry in describing the effects of spatial interactions between tree species on demographic rates. We also assess the congruence of these results with observed phylogenetic and functional structure in the neighbourhoods of live and dead trees. We found strong support for models in which the effects of spatial neighbourhood interactions on tree growth and survival were scaled to species-specific mean functional trait values (e.g., wood specific gravity, leaf succulence and maximum height) but not to phylogenetic distance. The weak phylogenetic signal in functional trait data allowed us to independently interpret the static neighbourhood functional and phylogenetic patterns. We observed greater functional trait similarity in the neighbourhoods of live trees relative to those of dead trees suggesting that environmental filtering is the major force structuring this tree community at this scale while competitive interactions play a lesser role.     

Identification, sequence determination, and expression of the flavodoxin gene from Desulfovibrio salexigens

Restriction fragments of genomic DNA from Desulfovibrio salexigens (ATCC 14822) containing the structural gene coding for the flavodoxin protein were identified using the entire coding region of the gene for the Desulfovibrio vulgaris (Hildenborough) flavodoxin as a probe (Krey, G.D., Vanin, E.F., and Swenson, R.P. (1988) J. Biol. Chem. 263, 15436-15443). A 1.4-kb PstI-HindIII fragment was ultimately identified which contains an open reading frame coding for a polypeptide of 146 amino acid residues that was highly homologous to the D. vulgaris flavodoxin, sharing a sequence identity of 55%. When compared to the X-ray crystal structure of the D. vulgaris protein, the homologous regions were largely confined to those portions of the protein which are in the immediate vicinity of the flavin mononucleotide cofactor binding site. Tryptophan-60 and tyrosine-98, which reside on either side of the isoalloxazine ring of the cofactor, are conserved, as are the sequences of the polypeptide loop that interacts with the phosphate moiety of the flavin. Acidic residues forming the interface of model electron-transfer complexes with certain cytochrome c proteins are retained. The flavodoxin holoprotein is over-expressed in E. coli from the cloned gene using its endogenous promoter.     

The effect of matrix on the occurrence of hazel grouse (Bonasa bonasia) in isolated habitat fragments

The aim of this study was to determine the effect of matrix on the occurrence of hazel grouse (Bonasa bonasia) in habitat fragments. The study was conducted in two kinds of landscape: (1) an agricultural landscape, where the censused forest habitat fragments were surrounded by farmland, and (2) in an intensively managed forested landscape, where the censused habitat fragments were surrounded by nonhabitat coniferous forest. Occupied and unoccupied habitat fragments in the agricultural landscape differed significantly in distance to the nearest suitable continuous habitat, with hazel grouse occurring only in habitat fragments closer than 100 m from continuous forest. In the intensively managed forest landscape, the effect of isolation was less evident, but there might be a threshold around 2 km. Effects of isolation occurred over much shorter distances when the surrounding habitats consisted of farmland than when it was forested habitats. The size of the habitat fragments was important in both landscapes, with larger habitat fragments more often containing hazel grouse.     

Testing the metabolic theory of ecology

The metabolic theory of ecology (MTE) predicts the effects of body size and temperature on metabolism through considerations of vascular distribution networks and biochemical kinetics. MTE has also been extended to characterise processes from cellular to global levels. MTE has generated both enthusiasm and controversy across a broad range of research areas. However, most efforts that claim to validate or invalidate MTE have focused on testing predictions. We argue that critical evaluation of MTE also requires strong tests of both its theoretical foundations and simplifying assumptions. To this end, we synthesise available information and find that MTE's original derivations require additional assumptions to obtain the full scope of attendant predictions. Moreover, although some of MTE's simplifying assumptions are well supported by data, others are inconsistent with empirical tests and even more remain untested. Further, although many predictions are empirically supported on average, work remains to explain the often large variability in data. We suggest that greater effort be focused on evaluating MTE's underlying theory and simplifying assumptions to help delineate the scope of MTE, generate new theory and shed light on fundamental aspects of biological form and function.     

Alveolar PCO2 oscillations and ventilation at sea level and at high altitude.

This study examines the potential for a ventilatory drive, independent of mean PCO2, but depending instead on changes in PCO2 that occur during the respiratory cycle. This responsiveness is referred to here as "dynamic ventilatory sensitivity." The normal, spontaneous, respiratory oscillations in alveolar PCO2 have been modified with inspiratory pulses approximating alveolar PCO2 concentrations, both at sea level and at high altitude (5,000 m, 16,400 ft.). All tests were conducted with subjects exercising on a cycle ergometer at 60 W. The pulses last about half the inspiratory duration and are timed to arrive in the alveoli during early or late inspiration. Differences in ventilation, which then occur in the face of similar end-tidal PCO2 values, are taken to result from dynamic ventilatory sensitivity. Highly significant ventilatory responses (early pulse response greater than late) occurred in hypoxia and normoxia at sea level and after more than 4 days at 5,000 m. The response at high altitude was eliminated by normalizing PO2 and was reduced or eliminated with acetazolamide. No response was present soon after arrival (<4 days) at base camp, 5,000 m, on either of two high-altitude expeditions (BMEME, 1994, and Kanchenjunga, 1998). The largest responses at 5,000 m were obtained in subjects returning from very high altitude (7,100-8,848 m). The present study confirms and extends previous investigations that suggest that alveolar PCO2 oscillations provide a feedback signal for respiratory control, independent of changes in mean PCO2, suggesting that natural PCO2 oscillations drive breathing in exercise.     

Should hunting mortality mimic the patterns of natural mortality?

With growing concerns about the impact of selective harvesting on natural populations, researchers encourage managers to implement harvest regimes that avoid or minimize the potential for demographic and evolutionary side effects. A seemingly intuitive recommendation is to implement harvest regimes that mimic natural mortality patterns. Using stochastic simulations based on a model of risk as a logistic function of a normally distributed biological trait variable, we evaluate the validity of this recommendation when the objective is to minimize the altering effect of harvest on the immediate post-mortality distribution of the trait. We show that, in the absence of compensatory mortality, harvest mimicking natural mortality leads to amplification of the biasing effect expected after natural mortality, whereas an unbiased harvest does not alter the post-mortality trait distribution that would be expected in the absence of harvest. Although our approach focuses only on a subset of many possible objectives for harvest management, it illustrates that a single strategy, such as hunting mimicking natural mortality, may be insufficient to address the complexities of different management objectives with potentially conflicting solutions.     

The role of evolutionary processes in producing biodiversity patterns, and the interrelationships between taxonomic, functional and phylogenetic biodiversity

Patterns of biodiversity are ultimately the product of speciation and extinction. Speciation serves as the biodiversity pump while extinction serves as the agent that culls global to local levels of biodiversity. Linking these central processes to global and local patterns of biodiversity is a key challenge in both ecology and evolution. This challenge necessarily requires a simultaneous consideration of the species, phylogenetic, and functional diversity across space and the tree of life. In this review, I outline a research framework for biodiversity science that considers the evolutionary and ecological processes that generate and cull levels of biodiversity and that influence the inter-relationships between species, phylogenetic, and functional diversity. I argue that a biodiversity synthesis must begin with a consideration of the inherently ecological process of speciation and end with how global biodiversity is filtered into local-scale plant communities. The review ends with a brief outlook on future challenges for those studying biodiversity, including outstanding hypotheses that need testing and key data limitations.