Conformational plasticity of proNGF

Nerve Growth Factor is an essential protein that supports neuronal survival during development and influences neuronal function throughout adulthood, both in the central and peripheral nervous system. The unprocessed precursor of NGF, proNGF, seems to be endowed with biological functions distinct from those of the mature protein, such as chaperone-like activities and apoptotic and/or neurotrophic properties. We have previously suggested, based on Small Angle X-ray Scattering data, that recombinant murine proNGF has features typical of an intrinsically unfolded protein. Using complementary biophysical techniques, we show here new evidence that clarifies and widens this hypothesis through a detailed comparison of the structural properties of NGF and proNGF. Our data provide direct information about the dynamic properties of the pro-peptide and indicate that proNGF assumes in solution a compact globular conformation. The N-terminal pro-peptide extension influences the chemical environment of the mature protein and protects the protein from proteolytic digestion. Accordingly, we observe that unfolding of proNGF involves a two-steps mechanism. The distinct structural properties of proNGF as compared to NGF agree with and rationalise a different functional role of the precursor.     

A kin-selection approach to the resolution of sex-ratio conflict between mates

We investigate an instance of conflict between mates over the sex ratio of their brood. We construct a kin-selection model for the evolution of the sex ratio assuming local resource competition (LRC) among females. We explore two basic scenarios: (a) the case where parents make simultaneous sex-ratio decisions (the simultaneous allocation model); and (b) the case where parental sex-ratio decisions occur one after the other (the sequential allocation model). In the simultaneous investment model, resolution of the conflict between mates depends on the extent to which relative paternal contribution influences the brood sex ratio. In the sequential allocation model, fathers determine primary sex-ratio through fertilization bias; then mothers modify the paternal sex-ratio decision by adjusting the level of investment of some resource that contributes to offspring survival. Under the sequential model, a compromise is always achieved; however this compromise favours one perspective or the other, depending on the extent to which maternal investment influences offspring survival.     

Long-term survival and concomitant gene expression of ribozyme-transduced CD4+ T-lymphocytes in HIV-infected patients

BACKGROUND: An anti-HIV-1 tat ribozyme, termed Rz2, has been shown to inhibit HIV-1 infection/replication and to decrease HIV-1-induced pathogenicity in T-lymphocyte cell lines and normal peripheral blood T-lymphocytes. We report here the results of a phase I gene transfer clinical trial using Rz2. METHODS: Apheresis was used to obtain a peripheral blood cell population from each of four HIV-negative donors. After enrichment for CD4+ T-lymphocytes, ex vivo expansion and genetic manipulation (approximately equal aliquots of the cells were transduced with the ribozyme-containing (RRz2) and the control (LNL6) retroviral vector), these cells were infused into the corresponding HIV-1-positive twin recipient. Marking was assessed over an initial 24-week period and in total over an approximate 4-year period. RESULTS: The gene transfer procedure was shown to be safe, and technically feasible. Both RRz2- and LNL6-gene-containing peripheral blood mononuclear cells (PBMC) were detected at all time points examined to 4 years. There was concomitant gene construct expression in the absence of the need for ex vivo peripheral blood cell stimulation and there was no evidence of immune elimination of the neoR T-lymphocytes nor of silencing of the Moloney murine leukemia virus long terminal repeat. CONCLUSIONS: The proof of principle results reported here demonstrate safety and feasibility of this type of gene transfer approach. While not specifically tested, T-lymphocytes containing an anti-HIV gene construct may impact on HIV-1 viral load and CD4+ T-lymphocyte count, potentially representing a new therapeutic modality for HIV-1 infection.     

Dynamics of Tree Diversity in Undisturbed and Logged Subtropical Rainforest in Australia

In subtropical rainforest in eastern Australia, changes in the diversity of trees were compared under natural conditions and eight silvicultural regimes over 35 years. In the treated plots basal area remaining after logging ranged from 12 to 58 m^{2}/ha. In three control plots richness differed little over this period. In the eight treated plots richness per plot generally declined after intervention and then gradually increased to greater than original diversity. After logging there was a reduction in richness per plot and an increase in species richness per stem in all but the lightest selective treatments. The change in species diversity was related to the intensity of the logging, however the time taken for species richness to return to pre-logging levels was similar in all silvicultural treatments and was not effected by the intensity of treatment. These results suggest that light selective logging in these forests mainly affects dominant species. The return to high diversity after only a short time under all silvicultural regimes suggests that sustainability and the manipulation of species composition for desired management outcomes is possible.     

Crystal lattice as biological phenotype for insect viruses

Many insect viruses survive for long periods by occlusion within robust crystalline polyhedra composed primarily of a single polyhedrin protein. We show that two different virus families form polyhedra which, despite lack of sequence similarity in the virally encoded polyhedrin protein, have identical cell constants and a body-centered cubic lattice. It is almost inconceivable that this could have arisen by chance, suggesting that the crystal lattice has been preserved because it is particularly well-suited to its function of packaging and protecting viruses.     

Chlortetracycline and demeclocycline inhibit calpains and protect mouse neurons against glutamate toxicity and cerebral ischemia

Minocycline is a potent neuroprotective tetracycline in animal models of cerebral ischemia. We examined the protective properties of chlortetracycline (CTC) and demeclocycline (DMC) and showed that these two tetracyclines were also potent neuroprotective against glutamate-induced neuronal death in vitro and cerebral ischemia in vivo. However, CTC and DMC appeared to confer neuroprotection through a unique mechanism compared with minocycline. Rather than inhibiting microglial activation and caspase, CTC and DMC suppressed calpain activities. In addition, CTC and DMC only weakly antagonized N-methyl-D-aspartate (NMDA) receptor activities causing 16 and 14%, respectively, inhibition of NMDA-induced whole cell currents and partially blocked NMDA-induced Ca2+ influx, commonly regarded as the major trigger of neuronal death. In vitro and in vivo experiments demonstrated that the two compounds selectively inhibited the activities of calpain I and II activated following glutamate treatment and cerebral ischemia. In contrast, minocycline did not significantly inhibit calpain activity. Taken together, these results suggested that CTC and DMC provide neuroprotection through suppression of a rise in intracellular Ca2+ and inhibition of calpains.     

Neurogenesis and neuronal communication on micropatterned neurochips

Neural networks are formed by accurate connectivity of neurons and glial cells in the brain. These networks employ a three-dimensional bio-surface that both assigns precise coordinates to cells during development and facilitates their connectivity and functionality throughout life. Using specific topographic and chemical features, we have taken steps towards the development of poly(dimethylsiloxane; PDMS) neurochips that can be used to generate and study synthetic neural networks. These neurochips have micropatterned structures that permit adequate cell positioning and support cell survival. Within days of plating, cells differentiate into neurons displaying excitability and communication, as evidenced by intracellular calcium oscillations and action potentials. The structural and functional capacities of such simple neural networks open up new opportunities to study synaptic communication and plasticity.     

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.     

Using stable isotopes to investigate migratory connectivity of the globally threatened aquatic warbler Acrocephalus paludicola

Understanding the links between breeding and wintering areas of migratory species has important ecological and conservation implications. Recently, stable isotope technology has been used to further our understanding. Stable isotope ratios vary geographically with a range of biogeochemical factors and isotope profiles in organisms reflect those in their food and environment. For inert tissues like feathers, isotope profiles reflect the environment in which they were formed. Following large-scale habitat destruction, the globally threatened aquatic warbler Acrocephalus paludicola has a fragmented breeding population across central Europe, largely in Belarus, Poland and Ukraine. The species sub-Saharan African wintering grounds have not yet been discovered, and this significantly hampers conservation efforts. Aquatic warblers grow their flight feathers on their wintering grounds, and we analysed stable isotope ratios (¹⁵N, ¹³C, D) in rectrices of adults from six main breeding sites (subpopulations) across Europe to determine whether different breeding subpopulations formed a single mixed population on the wintering grounds. ¹⁵N varies considerably with dietary trophic level and environmental factors, and D with the D in rainfall; neither varied between aquatic warbler subpopulations. Uniform feather ¹⁵N signatures suggest no major variation in dietary trophic level during feather formation. High variance and inter-annual differences in mean D values hinder interpretation of these data. Significant differences in mean ¹³C ratios existed between subpopulations. We discuss possible interpretations of this result, and consider differences in moulting latitude of different subpopulations to be the most parsimonious. ¹³C in plants and animals decreases with latitude, along a steep gradient in sub-Saharan Africa. Birds from the most north-westerly breeding subpopulation (Karsibor, Poland) had significantly lower variance in ¹³C and ¹⁵N than birds from all other sites, suggesting either that birds from Karsibor are less geographically dispersed during moult, or moult in an area with less isotopic heterogeneity. Mean ¹³C signatures from winter-grown feathers of different subpopulations were positively correlated with the latitude and longitude of breeding sites, suggesting a strong relationship between European breeding and African winter moulting latitudes. The use of stable isotopes provides novel insights into migratory connectivity and migration patterns in this little-known threatened species.