Detergent-insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1

Determining the composition of aggregated superoxide dismutase 1 (SOD1) species associated with amyotrophic lateral sclerosis (ALS), especially with respect to co-aggregated proteins and post-translational modifications, could identify cellular or biochemical factors involved in the formation of these aggregates and explain their apparent neurotoxicity. The results of mass spectrometric and shotgun-proteomic analyses of SOD1-containing aggregates isolated from spinal cords of symptomatic transgenic ALS mice using two different isolation strategies are presented, including 1) resistance to detergent extraction and 2) size exclusion-coupled anti-SOD1 immunoaffinity chromatography. Forty-eight spinal cords from three different ALS-SOD1 mutant mice were analyzed, namely G93A, G37R, and the unnatural double mutant H46R/H48Q. The analysis consistently revealed that the most abundant proteins recovered from aggregate species were full-length unmodified SOD1 polypeptides. Although aggregates from some spinal cord samples contained trace levels of highly abundant proteins, such as vimentin and neurofilament-3, no proteins were consistently found to co-purify with mutant SOD1 in stoichiometric quantities. The results demonstrate that the principal protein in the high molecular mass aggregates whose appearance correlates with symptoms of the disease is the unmodified, full-length SOD1 polypeptide.     

Closely related allopatric Podalyria species from the Core Cape Subregion differ in their mechanisms for acquisition of phosphorus,growth and ecological niche

Aims In the Core Cape Subregion (CCR), a Mediterranean-climate ecosystem with infertile soils, the legume species Podalyria calyptrata and P. burchellii are in a separate clade to P. leipoldtii and P. myrtillifolia. The closely related species are allopatric, and with the west-east climate gradient and variation in soil nutrient availability in the CCR, it was hypothesized that the two closely related allopatric species would differ in their ecological niche and root:shoot ratio, specific root length (SRL) and organic acid exudation responses to phosphorus (P) supply.Methods With increasing P supply in the glasshouse, we measured plant biomass, leaf nitrogen ([N]), [P], root morphology and release of organic acids. We determined species soil and leaf [N] and [P] and climate in field sites.Important findings At low P supply, P. calyptrata roots exuded more organic acids than P. burchellii which instead produced roots with a greater SRL, and P. myrtillifolia allocated more biomass to roots than P. leipoldtii. In the field, leaf [P] and climate suggested that P. leipoldtii occupied the most oligotrophic niche followed by P. burchellii and then P. calyptrata and P. myrtillifolia. Closely related allopatric species differed in their mechanisms for P-acquisition and ecological niche, indicating that the environment overrides phylogeny in determining P-acquisition traits for these species, and suggesting that climate regulates nutrient availability, driving distribution and speciation.     

Climate change, species range limits and body size in marine bivalves

We use data on the Pleistocene and modern range limits of Californian marine bivalves to show that species that shifted their geographical ranges in response to Pleistocene climatic fluctuations were preferentially drawn from the large end of the regional body size–frequency distributions. This difference is not due to phylogenetic effects (i.e. dominance of extralimital species by a few large-bodied clades), differences among major ecological categories (burrowing versus surface-dwelling, or suspension feeding versus non-suspension feeding), or differences in modes of reproduction and larval development. In addition, we show that successful invasive species of bivalves in present-day marine habitats also tend to be large-bodied, despite the difference in mechanisms between present-day and Pleistocene range expansions. These results indicate that range limits of large-bodied bivalve species are more unstable than small-bodied ones, and that body size and its correlates need to be considered when attempting to predict the responses of marine communities to climate change, biotic interchanges and human-mediated invasions.     

Optimal co-allocation of carbon and nitrogen in a forest stand at steady state

Nitrogen (N) is essential for plant production, but N uptake imposes carbon (C) costs through maintenance respiration and fine-root construction, suggesting that an optimal C:N balance can be found. Previous studies have elaborated this optimum under exponential growth; work on closed canopies has focused on foliage only. Here, the optimal co-allocation of C and N to foliage, fine roots and live wood is examined in a closed forest stand. Optimal co-allocation maximizes net primary productivity (NPP) as constrained by stand-level C and N balances and the pipe model. Photosynthesis and maintenance respiration increase with foliar nitrogen concentration ([N]), and stand-level photosynthesis and N uptake saturate at high foliage and fine-root density. Optimal NPP increases almost linearly from low to moderate N availability, saturating at high N. Where N availability is very low or very high, the system resembles a functional balance with a steady foliage [N]; in between, [N] increases with N availability. Carbon allocation to fine roots decreases, allocation to wood increases, and allocation to foliage remains stable with increasing N availability. The predicted relationships between biomass density and foliage [N] are in reasonable agreement with data from coniferous stands across Finland. All predictions agree with our qualitative understanding of N effects on growth.     

Relevance of studying T cell responses in SIV-infected rhesus macaques

HIV infection, once established, is never cleared. Rare individuals do, however, control viral replication to low levels. These successful immune responses are primarily linked to certain class I MHC alleles (MHC-I). Because of this association, many AIDS vaccines in development are designed to generate virus-specific CD8+ T cells. The Merck STEP phase 2b efficacy trial of one such vaccine was recently halted, and declared a failure. Thus, basic questions regarding what constitutes an effective T cell response and how such responses could be elicited by vaccination remain open. The best animal model available to explore such issues is simian immunodeficiency virus infection of rhesus macaques, which serves as the primary proving ground for AIDS vaccines.     

Lysophospholipid signaling: beyond the EDGs

As our understanding of the myriads of biological effects caused by lysophospholipids expands, we become witnesses to another miracle of nature that has endowed the simplest lysophospholipids with functions seemingly ubiquitous to every mammalian cell. A decade after the discovery of the EDG family lysophospholipid receptors, the field has gained unimaginable impetus explaining the biological effects of sphingosine-1-phosphate and lysophosphatidic acid (LPA). The discovery of LPA receptors in the purinergic G-protein-coupled receptor (GPCR) gene cluster refined this picture and added complexity to our concepts of lysophospholipid cell signaling. The intracellular lysophospholipid targets - identified and not yet identified - make us realize the dual mediator and second messenger roles of lysophospholipids. In this paper we provide new data obtained concerning LPA-elicited responses using cell lines naturally lacking or intentionally knocked out of many of the known LPA GPCR, widely used by investigators in the field as cells with LPA receptor "null background." Our observations raise caution about the lack of LPA responsiveness in these cells and underline the unprecedented complexity and redundancy of lysophospholipid-evoked cellular responses.     

Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.     

A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase.

The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity. The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif. Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved. Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC). Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure. The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking. Magnesium is required for primase-ssDNA binding. The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases. The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding. Some possible sources of this gain in entropy are discussed.     

Statistical and physiological distinction of constitution types

Most conventional human health and function evaluation methods are based on a traditional notion that all the population characteristics follow the Gaussian distribution law with the parameters M and s forming the basis of the norm conception. But some known facts contradict this idea that requires checking the statistical homogeneity of population characteristics. Analysis of statistical distribution and central tendencies for simple measured indices in population and somatotypes samples proved an idea of natural population distinctions by a broad set of morpho-functional features (by means of 23-D matrix cluster analysis for different indices) and provided the scientific grounds to use a constitutional approach in human sciences and physical education as well. Gaussian distribution law was found within somatotype groups permitting the use of its parameters for norm evaluation. In practice for somatotype determination the relative girth body dimensions (normalized by body height) were proved to be preferable.