Conformational effects of nucleotide exchange in ras p21 proteins as studied by fluorescence spectroscopy

AbstractThe intrinsic fluorescence properties of the oncogene protein p21N-ras, p21H-ras and one of its transforming mutants, p21N-ras (Va1112), have been investigated. A mutant containing a single tryptophan at position 28 in p21H-ras (Trp28) has been specifically engineered to provide a probe of protein conformation on nucleotide binding. The proteins produced essentially similar circular dichroism spectra typical of alpha/beta proteins. A decrease in the intensity of the fluorescence emission spectrum due to tyrosine occurred on GDP/GTP nucleotide exchange in the native and mutant proteins. Selective excitation of the single tryptophan in p21 produced a decrease in fluorescence intensity which was accompanied by a blue shift in the wavelength of maximum emission on nucleotide exchange. A reduction in the residual Mg2+ ion concentration enhanced this effect

Conformational properties of the prion octa-repeat and hydrophobic sequences

AbstractWe have used circular dichroism to study synthetic peptides from two important regions of the prion protein: the N-terminal octa-repeat domain and a highly conserved hydrophobic section. Our results show that the octa-repeat sequence in free solution can adopt a non-random, extended conformation with properties similar to the poly-l-proline type II left-handed helix. We also show that the conformation can be changed by temperature, organic solvents (e.g. acetonitrile) and on binding to phospholipid vesicles. We compared CD data from two peptides corresponding to the hydrophobic region between residues 106 and 136 which contained either methionine or valine at position 129. This variation represents a common polymorphism in humans which has been shown to influence predisposition towards iatrogenic and sporadic CJD. There was no detectable difference between the CD spectra of these peptides irrespective of the solvent conditions we used

The Extreme Hosts of Extreme Supernovae

We use GALEX ultraviolet (UV) and optical integrated photometry of the hosts of seventeen luminous supernovae (LSNe, having peak M_V < -21) and compare them to a sample of 26,000 galaxies from a cross-match between the SDSS DR4 spectral catalog and GALEX interim release 1.1. We place the LSNe hosts on the galaxy NUV-r versus M_r color magnitude diagram (CMD) with the larger sample to illustrate how extreme they are. The LSN hosts appear to favor low-density regions of the galaxy CMD falling on the blue edge of the blue cloud toward the low luminosity end. From the UV-optical photometry, we estimate the star formation history of the LSN hosts. The hosts have moderately low star formation rates (SFRs) and low stellar masses (M_*) resulting in high specific star formation rates (sSFR). Compared with the larger sample, the LSN hosts occupy low-density regions of a diagram plotting sSFR versus M_* in the area having higher sSFR and lower M_*. This preference for low M_*, high sSFR hosts implies the LSNe are produced by an effect having to do with their local environment. The correlation of mass with metallicity suggests that perhaps wind-driven mass loss is the factor that prevents LSNe from arising in higher-mass, higher-metallicity hosts. The massive progenitors of the LSNe (>100 M_sun), by appearing in low-SFR hosts, are potential tests for theories of the initial mass function that limit the maximum mass of a star based on the SFR.Comment: 8 pages, 3 figures, 2 tables, accepted to ApJ, amended references and updated SN designation

A pleurocidin analogue with greater conformational flexibility, enhanced antimicrobial potency and in vivo therapeutic efficacy.

Antimicrobial peptides (AMPs) are a potential alternative to classical antibiotics that are yet to achieve a therapeutic breakthrough for treatment of systemic infections. The antibacterial potency of pleurocidin, an AMP from Winter Flounder, is linked to its ability to cross bacterial plasma membranes and seek intracellular targets while also causing membrane damage. Here we describe modification strategies that generate pleurocidin analogues with substantially improved, broad spectrum, antibacterial properties, which are effective in murine models of bacterial lung infection. Increasing peptide-lipid intermolecular hydrogen bonding capabilities enhances conformational flexibility, associated with membrane translocation, but also membrane damage and potency, most notably against Gram-positive bacteria. This negates their ability to metabolically adapt to the AMP threat. An analogue comprising D-amino acids was well tolerated at an intravenous dose of 15 mg/kg and similarly effective as vancomycin in reducing EMRSA-15 lung CFU. This highlights the therapeutic potential of systemically delivered, bactericidal AMPs

Theory and Applications of Non-Relativistic and Relativistic Turbulent Reconnection

Realistic astrophysical environments are turbulent due to the extremely high Reynolds numbers. Therefore, the theories of reconnection intended for describing astrophysical reconnection should not ignore the effects of turbulence on magnetic reconnection. Turbulence is known to change the nature of many physical processes dramatically and in this review we claim that magnetic reconnection is not an exception. We stress that not only astrophysical turbulence is ubiquitous, but also magnetic reconnection itself induces turbulence. Thus turbulence must be accounted for in any realistic astrophysical reconnection setup. We argue that due to the similarities of MHD turbulence in relativistic and non-relativistic cases the theory of magnetic reconnection developed for the non-relativistic case can be extended to the relativistic case and we provide numerical simulations that support this conjecture. We also provide quantitative comparisons of the theoretical predictions and results of numerical experiments, including the situations when turbulent reconnection is self-driven, i.e. the turbulence in the system is generated by the reconnection process itself. We show how turbulent reconnection entails the violation of magnetic flux freezing, the conclusion that has really far reaching consequences for many realistically turbulent astrophysical environments. In addition, we consider observational testing of turbulent reconnection as well as numerous implications of the theory. The former includes the Sun and solar wind reconnection, while the latter include the process of reconnection diffusion induced by turbulent reconnection, the acceleration of energetic particles, bursts of turbulent reconnection related to black hole sources as well as gamma ray bursts. Finally, we explain why turbulent reconnection cannot be explained by turbulent resistivity or derived through the mean field approach.Comment: 66 pages, 24 figures, a chapter of the book "Magnetic Reconnection - Concepts and Applications", editors W. Gonzalez, E. N. Parke

Multivariate Computational Analysis of Gamma Delta T Cell Inhibitory Receptor Signatures Reveals the Divergence of Healthy and ART-Suppressed HIV+ Aging

Even with effective viral control, HIV-infected individuals are at a higher risk for morbidities associated with older age than the general population, and these serious non-AIDS events (SNAEs) track with plasma inflammatory and coagulation markers. The cell subsets driving inflammation in aviremic HIV infection are not yet elucidated. Also, whether ART-suppressed HIV infection causes premature induction of the inflammatory events found in uninfected elderly or if a novel inflammatory network ensues when HIV and older age co-exist is unclear. In this study we measured combinational expression of five inhibitory receptors (IRs) on seven immune cell subsets and 16 plasma markers from peripheral blood mononuclear cells (PBMC) and plasma samples, respectively, from a HIV and Aging cohort comprised of ART-suppressed HIV-infected and uninfected controls stratified by age (≤35 or ≥50 years old). For data analysis, multiple multivariate computational algorithms [cluster identification, characterization, and regression (CITRUS), partial least squares regression (PLSR), and partial least squares-discriminant analysis (PLS-DA)] were used to determine if immune parameter disparities can distinguish the subject groups and to investigate if there is a cross-impact of aviremic HIV and age on immune signatures. IR expression on gamma delta (γδ) T cells exclusively separated HIV+ subjects from controls in CITRUS analyses and secretion of inflammatory cytokines and cytotoxic mediators from γδ T cells tracked with TIGIT expression among HIV+ subjects. Also, plasma markers predicted the percentages of TIGIT+ γδ T cells in subjects with and without HIV in PSLR models, and a PLS-DA model of γδ T cell IR signatures and plasma markers significantly stratified all four of the subject groups (uninfected younger, uninfected older, HIV+ younger, and HIV+ older). These data implicate γδ T cells as an inflammatory driver in ART-suppressed HIV infection and provide evidence of distinct “inflamm-aging” processes with and without ART-suppressed HIV infection

Incompatibilities Involving Yeast Mismatch Repair Genes: A Role for Genetic Modifiers and Implications for Disease Penetrance and Variation in Genomic Mutation Rates

Genetic background effects underlie the penetrance of most genetically determined phenotypes, including human diseases. To explore how such effects can modify a mutant phenotype in a genetically tractable system, we examined an incompatibility involving the MLH1 and PMS1 mismatch repair genes using a large population sample of geographically and ecologically diverse Saccharomyces cerevisiae strains. The mismatch repair incompatibility segregates into naturally occurring yeast strains, with no strain bearing the deleterious combination. In assays measuring the mutator phenotype conferred by different combinations of MLH1 and PMS1 from these strains, we observed a mutator phenotype only in combinations predicted to be incompatible. Surprisingly, intragenic modifiers could be mapped that specifically altered the strength of the incompatibility over a 20-fold range. Together, these observations provide a powerful model in which to understand the basis of disease penetrance and how such genetic variation, created through mating, could result in new mutations that could be the raw material of adaptive evolution in yeast populations

Analysis of the interaction with the hepatitis C virus mRNA reveals an alternative mode of RNA recognition by the human La protein

Human La protein is an essential factor in the biology of both coding and non-coding RNAs. In the nucleus, La binds primarily to 3′ oligoU containing RNAs, while in the cytoplasm La interacts with an array of different mRNAs lacking a 3′ UUUOH trailer. An example of the latter is the binding of La to the IRES domain IV of the hepatitis C virus (HCV) RNA, which is associated with viral translation stimulation. By systematic biophysical investigations, we have found that La binds to domain IV using an RNA recognition that is quite distinct from its mode of binding to RNAs with a 3′ UUUOH trailer: although the La motif and first RNA recognition motif (RRM1) are sufficient for high-affinity binding to 3′ oligoU, recognition of HCV domain IV requires the La motif and RRM1 to work in concert with the atypical RRM2 which has not previously been shown to have a significant role in RNA binding. This new mode of binding does not appear sequence specific, but recognizes structural features of the RNA, in particular a double-stranded stem flanked by single-stranded extensions. These findings pave the way for a better understanding of the role of La in viral translation initiation

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

Heterodimerization of the human RNase P/MRP subunits Rpp20 and Rpp25 is a prerequisite for interaction with the P3 arm of RNase MRP RNA

Rpp20 and Rpp25 are two key subunits of the human endoribonucleases RNase P and MRP. Formation of an Rpp20–Rpp25 complex is critical for enzyme function and sub-cellular localization. We present the first detailed in vitro analysis of their conformational properties, and a biochemical and biophysical characterization of their mutual interaction and RNA recognition. This study specifically examines the role of the Rpp20/Rpp25 association in the formation of the ribonucleoprotein complex. The interaction of the individual subunits with the P3 arm of the RNase MRP RNA is revealed to be negligible whereas the 1:1 Rpp20:Rpp25 complex binds to the same target with an affinity of the order of nM. These results unambiguously demonstrate that Rpp20 and Rpp25 interact with the P3 RNA as a heterodimer, which is formed prior to RNA binding. This creates a platform for the design of future experiments aimed at a better understanding of the function and organization of RNase P and MRP. Finally, analyses of interactions with deletion mutant proteins constructed with successively shorter N- and C-terminal sequences indicate that the Alba-type core domain of both Rpp20 and Rpp25 contains most of the determinants for mutual association and P3 RNA recognition