Molecular characterization of an anion pump. The ArsB protein is the membrane anchor for the ArsA protein

R-factor mediated bacterial resistance to arsenical salts occurs by active extrusion of the toxic oxyanions from cells of gram negative bacteria. The ars operon of the conjugative plasmid R773 encodes an anion pump. The pump has two polypeptide components. The catalytic subunit, the ArsA protein, is an oxyanion-stimulated ATPase. The membrane component, the ArsB protein, has been localized in the inner membrane of Escherichia coli. The ArsA and ArsB proteins have been postulated to form a membrane complex which functions as an anion-translocating ATPase. In this study evidence is presented showing that expression of the arsB gene is required to anchor the ArsA protein to the inner membrane. Binding studies with purified ArsA to membranes with and without the arsB gene product confirm this requirement. Membranes of uncA mutants containing both the ArsA and ArsB proteins exhibit arsenite(antimonite)-stimulated ATPase activity. These results support the model in which the ArsA protein is the catalytic energy transducing component of the anion pump, whereas the integral membrane ArsB protein serves as both the anion channel and membrane binding site for the ArsA protein

Nonet Symmetry and Two-Body Decays of Charmed Mesons

The decay of charmed mesons into pseudoscalar (P) and vector (V) mesons is studied in the context of nonet symmetry. We have found that it is badly broken in the PP channels and in the P sector of the PV channels as expected from the non-ideal mixing of the \eta and the \eta'. In the VV channels, it is also found that nonet symmetry does not describe the data well. We have found that this discrepancy cannot be attributed entirely to SU(3) breaking at the usual level of 20--30%. At least one, or both, of nonet and SU(3) symmetry must be very badly broken. The possibility of resolving the problem in the future is also discussed.Comment: 9 pages, UTAPHY-HEP-

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed

Contributions of temporal encodings of voicing, voicelessness, fundamental frequency, and amplitude variation to audiovisual and auditory speech perception

Auditory and audio-visual speech perception was investigated using auditory signals of invariant spectral envelope that temporally encoded the presence of voiced and voiceless excitation, variations in amplitude envelope and F-0. In experiment 1, the contribution of the timing of voicing was compared in consonant identification to the additional effects of variations in F-0 and the amplitude of voiced speech. In audio-visual conditions only, amplitude variation slightly increased accuracy globally and for manner features. F-0 variation slightly increased overall accuracy and manner perception in auditory and audio-visual conditions. Experiment 2 examined consonant information derived from the presence and amplitude variation of voiceless speech in addition to that from voicing, F-0, and voiced speech amplitude. Binary indication of voiceless excitation improved accuracy overall and for voicing and manner. The amplitude variation of voiceless speech produced only a small increment in place of articulation scores. A final experiment examined audio-visual sentence perception using encodings of voiceless excitation and amplitude variation added to a signal representing voicing and F-0. There was a contribution of amplitude variation to sentence perception, but not of voiceless excitation. The timing of voiced and voiceless excitation appears to be the major temporal cues to consonant identity. (C) 1999 Acoustical Society of America. [S0001-4966(99)01410-1]

A survey of stellar X-ray flares from the XMM-Newton serendipitous source catalogue: Hipparcos-Tycho cool stars

The X-ray emission from flares on cool (i.e. spectral-type F-M) stars is indicative of very energetic, transient phenomena, associated with energy release via magnetic reconnection. We present a uniform, large-scale survey of X-ray flare emission. The XMM-Newton Serendipitous Source Catalogue and its associated data products provide an excellent basis for a comprehensive and sensitive survey of stellar flares - both from targeted active stars and from those observed serendipitously in the half-degree diameter field-of-view of each observation. The 2XMM Catalogue and the associated time-series (`light-curve') data products have been used as the basis for a survey of X-ray flares from cool stars in the Hipparcos Tycho-2 catalogue. In addition, we have generated and analysed spectrally-resolved (i.e. hardness-ratio), X-ray light-curves. Where available, we have compared XMM OM UV/optical data with the X-ray light-curves. Our sample contains ~130 flares with well-observed profiles; they originate from ~70 stars. The flares range in duration from ~1e3 to ~1e4 s, have peak X-ray fluxes from ~1e-13 to ~1e-11 erg/cm2/s, peak X-ray luminosities from ~1e29 to ~1e32 erg/s, and X-ray energy output from ~1e32 to ~1e35 erg. Most of the ~30 serendipitously-observed stars have little previously reported information. The hardness-ratio plots clearly illustrate the spectral (and hence inferred temperature) variations characteristic of many flares, and provide an easily accessible overview of the data. We present flare frequency distributions from both target and serendipitous observations. The latter provide an unbiased (with respect to stellar activity) study of flare energetics; in addition, they allow us to predict numbers of stellar flares that may be detected in future X-ray wide-field surveys. The serendipitous sample demonstrates the need for care when calculating flaring rates.Comment: 26 pages, 24 figures. Additional tables and figures available as 4 ancillary files. To be published in Astronomy and Astrophysic

Pathway of human AS3MT arsenic methylation

A synthetic gene encoding human As(III) S-adenosylmethionine (SAM) methyltransferase (hAS3MT) was expressed, and the purified enzyme was characterized. The synthetic enzyme is considerably more active than a cDNA-expressed enzyme using endogenous reductants thioredoxin (Trx), thioredoxin reductase (TR), NADPH, and reduced glutathione (GSH). Each of the seven cysteines (the four conserved residues, Cys32, Cys61, Cys156, and Cys206, and nonconserved, Cys72, Cys85, and Cys250) was individually changed to serine. The nonconserved cysteine derivates were still active. None of the individual C32S, C61S, C156S, and C206S derivates were able to methylate As(III). However, the C32S and C61S enzymes retained the ability to methylate MAs(III). These observations suggest that Cys156 and Cys206 play a different role in catalysis than that of Cys32 and Cys61. A homology model built on the structure of a thermophilic orthologue indicates that Cys156 and Cys206 form the As(III) binding site, whereas Cys32 and Cys61 form a disulfide bond. Two observations shed light on the pathway of methylation. First, binding assays using the fluorescence of a single-tryptophan derivative indicate that As(GS)3 binds to the enzyme much faster than inorganic As(III). Second, the major product of the first round of methylation is MAs(III), not MAs(V), and remains enzyme-bound until it is methylated a second time. We propose a new pathway for hAS3MT catalysis that reconciles the hypothesis of Challenger ((1947) Sci. Prog., 35, 396-416) with the pathway proposed by Hayakawa et al. ((2005) Arch. Toxicol., 79, 183-191). The products are the more toxic and more carcinogenic trivalent methylarsenicals, but arsenic undergoes oxidation and reduction as enzyme-bound intermediates