Influenza B virus BM2 protein is an oligomeric integral membrane protein expressed at the cell surface

AbstractThe influenza B virus BM2 protein contains 109 amino acid residues and it is translated from a bicistronic mRNA in an open reading frame that is +2 nucleotides with respect to the matrix (M1) protein. The amino acid sequence of BM2 contains a hydrophobic region (residues 7–25) that could act as a transmembrane (TM) anchor. Analysis of properties of the BM2 protein, including detergent solubility, insolubility in alkali pH 11, flotation in membrane fractions, and epitope-tagging immunocytochemistry, indicates BM2 protein is the fourth integral membrane protein encoded by influenza B virus in addition to hemagglutinin (HA), neuraminidase (NA), and the NB glycoprotein. Biochemical analysis indicates that the BM2 protein adopts an NoutCin orientation in membranes and fluorescence microscopy indicates BM2 is expressed at the cell surface. As the BM2 protein possesses only a single hydrophobic domain and lacks a cleavable signal sequence, it is another example of a Type III integral membrane protein, in addition to M2, NB, and CM2 proteins of influenza A, B, and C viruses, respectively. Chemical cross-linking studies indicate that the BM2 protein is oligomeric, most likely a tetramer. Comparison of the amino acid sequence of the TM domain of the BM2 protein with the sequence of the TM domain of the proton-selective ion channel M2 protein of influenza A virus is intriguing as M2 protein residues critical for ion selectivity/activation and channel gating (H37 and W41, respectively) are found at the same relative position and spacing in the BM2 protein (H19 and W23)

Discovery of Spiro-Piperidine Inhibitors and Their Modulation of the Dynamics of the M2 Proton Channel from Influenza A Virus

Amantadine has been used for decades as an inhibitor of the influenza A virus M2 protein (AM2) in the prophylaxis and treatment of influenza A infections, but its clinical use has been limited by its central nervous system (CNS) side effects as well as emerging drug-resistant strains of the virus. With the goal of searching for new classes of M2 inhibitors, a structure−activity relation study based on 2-[3-azaspiro(5,5)undecanol]-2-imidazoline (BL-1743) was initiated. The first generation BL-1743 series of compounds has been synthesized and tested by two-electrode voltage-clamp (TEV) assays. The most active compound from this library, 3-azaspiro[5,5]undecane hydrochloride (9), showed an IC50 as low as 0.92 ± 0.11 μM against AM2, more than an order of magnitude more potent than amantadine (IC50 = 16 μM). 15N and 13C solid-state NMR was employed to determine the effect of compound 9 on the structure and dynamics of the transmembrane domain of AM2 (AM2-TM) in phospholipid bilayers. Compared to amantadine, spiro-piperidine 9 (1) induces a more homogeneous conformation of the peptide, (2) reduces the dynamic disorder of the G34-I35 backbone near the water-filled central cavity of the helical bundle, and (3) influences the dynamics and magnetic environment of more residues within the transmembrane helices. These data suggest that spiro-piperidine 9 binds more extensively with the AM2 channel, thus leading to stronger inhibitory potency

Genetic Interactions between Chromosomes 11 and 18 Contribute to Airway Hyperresponsiveness in Mice

We used two-dimensional quantitative trait locus analysis to identify interacting genetic loci that contribute to the native airway constrictor hyperresponsiveness to methacholine that characterizes A/J mice, relative to C57BL/6J mice. We quantified airway responsiveness to intravenous methacholine boluses in eighty-eight (C57BL/6J X A/J) F2 and twenty-seven (A/J X C57BL/6J) F2 mice as well as ten A/J mice and six C57BL/6J mice; all studies were performed in male mice. Mice were genotyped at 384 SNP markers, and from these data two-QTL analyses disclosed one pair of interacting loci on chromosomes 11 and 18; the homozygous A/J genotype at each locus constituted the genetic interaction linked to the hyperresponsive A/J phenotype. Bioinformatic network analysis of potential interactions among proteins encoded by genes in the linked regions disclosed two high priority subnetworks - Myl7, Rock1, Limk2; and Npc1, Npc1l1. Evidence in the literature supports the possibility that either or both networks could contribute to the regulation of airway constrictor responsiveness. Together, these results should stimulate evaluation of the genetic contribution of these networks in the regulation of airway responsiveness in humans.</p

GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion mutation within intron 1 of the FXN gene. However, the origins of the GAA repeat expansion, its unstable dynamics within different cells and tissues, and its effects on frataxin expression are not yet completely understood. Therefore, we have chosen to generate representative FRDA mouse models by using the human FXN GAA repeat expansion itself as the genetically modified mutation. We have previously reported the establishment of two lines of human FXN YAC transgenic mice that contain unstable GAA repeat expansions within the appropriate genomic context. We now describe the generation of FRDA mouse models by crossbreeding of both lines of human FXN YAC transgenic mice with heterozygous Fxn knockout mice. The resultant FRDA mice that express only human-derived frataxin show comparatively reduced levels of frataxin mRNA and protein expression, decreased aconitase activity, and oxidative stress, leading to progressive neurodegenerative and cardiac pathological phenotypes. Coordination deficits are present, as measured by accelerating rotarod analysis, together with a progressive decrease in locomotor activity and increase in weight. Large vacuoles are detected within neurons of the dorsal root ganglia (DRG), predominantly within the lumbar regions in 6-month-old mice, but spreading to the cervical regions after 1 year of age. Secondary demyelination of large axons is also detected within the lumbar roots of older mice. Lipofuscin deposition is increased in both DRG neurons and cardiomyocytes, and iron deposition is detected in cardiomyocytes after 1 year of age. These mice represent the first GAA repeat expansion-based FRDA mouse models that exhibit progressive FRDA-like pathology and thus will be of use in testing potential therapeutic strategies, particularly GAA repeat-based strategies. (c) 2006 Elsevier Inc. All rights reserved

Localization and characterization of somatostatin binding sites in the mouse retina

We studied the binding of [125I]Tyr11-somatostatin-14 and [125I]Leu8,-Trp22, Tyr25-somatostatin-28 to frozen, unfixed sections of C57BL/6J mouse eyes with autoradiography. Specific binding of both ligands occurred in 3 maxima, a broad band extending from the retinal ganglion cell to the inner nuclear layers, a narrow and inconstant band over the outer plexiform layer, and a band over the retinal pigment epithelium and choroid. We quantified the label over the inner plexiform layer and found evidence for a single, saturable binding site after Scatchard analysis of saturation binding data. With [125I]Tyr11-somatostatin-14 the dissociation constant (Kd) was 1.48 nM and the total number of binding sites (Bmax) was 68 fmol/mg protein; in competition experiments the inhibitory binding constant (Ki) was 900 pM for somatostatin-14 and 350 pM for somatostatin-28. With [125I]Leu8,-Trp22, Tyr25-somatostatin-28, Kd was 625 pM and Bmax was 69 fmol/mg protein: in competition experiments Ki was 4.58 nM for somatostatin-14 and 710 pM for somatostatin-28. These results demonstrate the existence of somatostatin receptors in the inner plexiform layer of the retina that appear to have greater specificity for somatostatin-28 than for somatostatin-14.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28093/1/0000540.pd

The photometric properties of a vast stellar substructure in the outskirts of M33

We have surveyed $\sim40$sq.degrees surrounding M33 with CFHT MegaCam in the g and i filters, as part of the Pan-Andromeda Archaeological Survey. Our observations are deep enough to resolve the top 4mags of the red giant branch population in this galaxy. We have previously shown that the disk of M33 is surrounded by a large, irregular, low-surface brightness substructure. Here, we quantify the stellar populations and structure of this feature using the PAndAS data. We show that the stellar populations of this feature are consistent with an old population with $\sim-1.6$dex and an interquartile range in metallicity of $\sim0.5$dex. We construct a surface brightness map of M33 that traces this feature to $\mu_V\simeq33$mags\,arcsec$^{-2}$. At these low surface brightness levels, the structure extends to projected radii of $\sim40$kpc from the center of M33 in both the north-west and south-east quadrants of the galaxy. Overall, the structure has an "S-shaped" appearance that broadly aligns with the orientation of the HI disk warp. We calculate a lower limit to the integrated luminosity of the structure of $-12.7\pm0.5$mags, comparable to a bright dwarf galaxy such as Fornax or AndII and slightly less than $1\$ of the total luminosity of M33. Further, we show that there is tentative evidence for a distortion in the distribution of young stars near the edge of the HI disk that occurs at similar azimuth to the warp in HI. The data also hint at a low-level, extended stellar component at larger radius that may be a M33 halo component. We revisit studies of M33 and its stellar populations in light of these new results, and we discuss possible formation scenarios for the vast stellar structure. Our favored model is that of the tidal disruption of M33 in its orbit around M31.Comment: Accepted for publication in ApJ. 17 figures. ApJ preprint forma

GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology

Friedreich ataxia (FRDA) is a neurodegenerative disorder caused by an unstable GAA repeat expansion mutation within intron 1 of the FXN gene. However, the origins of the GAA repeat expansion, its unstable dynamics within different cells and tissues, and its effects on frataxin expression are not yet completely understood. Therefore, we have chosen to generate representative FRDA mouse models by using the human FXN GAA repeat expansion itself as the genetically modified mutation. We have previously reported the establishment of two lines of human FXN YAC transgenic mice that contain unstable GAA repeat expansions within the appropriate genomic context. We now describe the generation of FRDA mouse models by crossbreeding of both lines of human FXN YAC transgenic mice with heterozygous Fxn knockout mice. The resultant FRDA mice that express only human-derived frataxin show comparatively reduced levels of frataxin mRNA and protein expression, decreased aconitase activity, and oxidative stress, leading to progressive neurodegenerative and cardiac pathological phenotypes. Coordination deficits are present, as measured by accelerating rotarod analysis, together with a progressive decrease in locomotor activity and increase in weight. Large vacuoles are detected within neurons of the dorsal root ganglia (DRG), predominantly within the lumbar regions in 6-month-old mice, but spreading to the cervical regions after 1 year of age. Secondary demyelination of large axons is also detected within the lumbar roots of older mice. Lipofuscin deposition is increased in both DRG neurons and cardiomyocytes, and iron deposition is detected in cardiomyocytes after 1 year of age. These mice represent the first GAA repeat expansion-based FRDA mouse models that exhibit progressive FRDA-like pathology and thus will be of use in testing potential therapeutic strategies, particularly GAA repeat-based strategies. © 2006 Elsevier Inc. All rights reserved

Southern Infrared Proper Motion Survey III: Constraining the mass function of low mass stars

The stellar mass function is one of the fundamental distributions of stellar astrophysics. Its form at masses similar to the Sun was found by Salpeter (1955) to be a power-law $m^{-\alpha}$ with a slope of $\alpha=1.35$. Since then the mass function in the field, in stellar clusters and in other galaxies has been studied to identify variation due to environment and mass range. Here we use results from previous papers in the SIPS series to constrain the mass function of low mass stars (0.075M$_\odot$$<$m$<0.2_\odot$). We use simulations of the low mass local stellar population based on those in Deacon & Hambly (2006) to model the results of the SIPS-II survey (Deacon & Hambly, 2007). We then vary the input parameters of these simulations (the exponent of the mass function $\alpha$ and a stellar birthrate parameter $\beta$) and compare the simulated survey results with those from the actual survey. After a correction for binarity and taking into account potential errors in our model we find that $\alpha=-0.62\pm0.26$ for the quoted mass range.Comment: Submitted to astro-p