In vivo peroxidative activity of FALS-mutant human CuZnSODs expressed in yeast.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder leading to loss of motor neurons. We previously characterized the enhanced peroxidative activity of the human familial ALS (FALS) mutants of copper-zinc superoxide dismutase (CuZnSOD) A4V and G93A in vitro. Here, a similar activity is demonstrated for human FALS CuZnSOD mutants in an in vivo model system, the yeast Saccharomyces cerevisiae. Spin trap adducts of alpha-(pyridyl-4-N-oxide)-N-tert-butylnitrone (POBN) have been measured by electron paramagnetic resonance (EPR) in yeast expressing mutant (A4V, L38V, G93A, and G93C) and wild type CuZnSOD upon addition of hydrogen peroxide to the culture. The trapped radical is a hydroxyethyl adduct of POBN, identified by spectral parameters. Mutant CuZnSODs produced greater concentrations of the trapped adduct compared to the wild type enzyme. This observation provides evidence for an oxidative radical mechanism, whereby the mutants of CuZnSOD catalyze the formation of reactive oxygen species that may be related to the development or progression of FALS. This study also presents an in vivo model system to study free radical production in FALS-associated CuZnSOD mutations.     

One-kilobase direct repeats of plasmid pSa

One-kilobase, direct repeats were found on either side of the chloramphenicol resistance gene of plasmid pSa. The right repeat corresponded to the region coding for sulfanilamide resistance. The repeats were not identical as judged by distances between restriction enzyme sites, hybridization, and by the ability to confer resistance to sulfanilamide.     

Purification and characterization of cardiac tropomyosins.

A new procedure was developed to purify tropomyosin. The procedure was an adaptation of that described for purification of myosin. By eliminating troponin before precipitating with (NH4)2 SO4, it was possible to obtain pure tropomyosin from the same preparation from which myosin was purified. When tropomyosin was subjected to isoelectrofocusing two tropomyosins were present, having similar isoelectric points of pH 5.4 and 5.6; two tropomyosin subunits were resolved in the presence of 6 M urea. The two subunits had very similar isoelectric points, pH 4.7 and 5.0. According to Ouchterlony analyses the tropomyosins from canine skeletal and cardiac tissue were immunologically identical when incubated with goat gammaG antitropomyosin (cardiac).     

Dynamics of fd coat protein in the bacteriophage

The dynamics of the coat protein in fd bacteriophage are described with solid-state 15N and 2H NMR experiments. The virus particles and the coat protein subunits are immobile on the time scales of the 15N chemical shift anisotropy (10(3) Hz) and 2H quadrupole (10(6) Hz) interactions. Previously we have shown that the Trp-26 side chain is immobile, that the two Tyr and three Phe side chains undergo only rapid twofold jump motions about their C beta-C gamma bond axis [Gall, C. M., Cross, T. A., DiVerdi, J. A., & Opella, S. J. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 101-105], and that most of the backbone peptide linkages are highly constrained but do undergo rapid small amplitude motions [Cross, T. A., & Opella, S. J. (1982) J. Mol. Biol. 159, 543-549] in the coat protein subunits in the virus particles. In this paper, we demonstrate that the four N-terminal residues of the coat protein subunits are highly mobile, since both backbone and side-chain sites of these residues undergo large amplitude motions that are rapid on the time scales of the solid-state NMR experiments. In addition, the dynamics of the methyl-containing aliphatic residues Ala, Leu, Val, Thr, and Met are analyzed. Large amplitude jump motions are observed in nearly all of these side chains even though, with the exception of the N-terminal residue Ala-1, their backbone peptide linkages are highly constrained. The established information about the dynamics of the structural form of fd coat protein in the virus particle is summarized qualitatively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Benzodiazepine receptor mediated discriminative cues: Effects of GABA-ergic drugs and inverse agonists

Rats were exposed to a two-layer drug discrimination procedure using the benzodiazepine (BZ) receptor inverse agonists N′-methyl-β-carboline-3-carboxamide (FG 7142) or methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM). FG 7142 (30 mg/kg) failed to acquire discriminative stimulus control, although it did suppress responding. The same group of animals was trained successfully to discriminate diazepam (DZP, 2.5 mg/kg) from vehicle. The DZP cue was potentiated by the GABA agonist 4,5,6,7-tetrahydro-isoxazolo [5, 4-c] pyridin-3-ol (THIP, 1–3 mg/kg); THIP alone produced vehicle-appropriate responding. In addition, clonazepam (0.2 mg/kg) and chlordiazepoxide (5 mg/kg) substituted for DZP (with potencies of 7.5 and 0.25 times that of DZP, respectively). In antagonism tests, FG 7142 (5–17.5 mg/kg), methyl-β-carboline-3-carboxylate (β-CCM, 2.5 mg/kg), nicotine (0.3 mg/kg), harmaline (5 mg/kg) and naltrexone (10 mg/kg) did not effect, bicuculine (2 mg/kg) and DMCM (1 mg/kg) partially blocked, and the BZ receptor antagonist Ro 15–1788 (40 mg/kg) completely blocked the discriminative stimulus effects of DZP. In animals trained to discriminate DMCM (0.2 mg/kg) from vehicle, 95% substitution occured with bicuculline (2 mg/kg); DZP (1–5 mg/kg) completely antagonized DMCM. These results indicate that the DZP cue is mediated by GABA-coupled BZ receptors and that GABA may modulate the efficacy of a BZ at its receptor site. However, since inverse BZ receptor agonists (FG 7142, DMCM and β-CCM) were, at best, only marginally effective in antagonizing DZP, the DZP cue may be mediated by a distinct subclass of BZ receptors.     

Demographic responses of an arboreal marsupial,the common brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>), to a prescribed fire

We investigated demographic responses of the common brushtail possum Trichosurus vulpecula, a medium-sized arboreal marsupial, after a prescribed fuel reduction burn on Magnetic Island, tropical north Queensland, Australia. Possums were live-trapped every month for 14 months before the fire and 11 months after the fire in both the burnt and unburnt areas; measurements of individuals were taken each month and demographic parameters were modelled using capture–mark–recapture data. Significant differences between the burnt and unburnt sites were found following the fire; recruitment was lower in the unburnt area, where population size also declined. In the burnt area, population size and recruitment displayed a tendency to increase after the fire, while capture probability declined, suggesting that an influx of new individuals, attracted to re-sprouting vegetation, had resulted in trap saturation. There was no detectable effect of the fire on survival, and no fire-induced mortalities were observed. We conclude that a low-intensity, prescribed, fuel-reduction burn had no obvious negative consequences for this possum population.     

Temperature sensitive nitrogen fixation mutants of Azotobacter vinelandii

Summary Temperature-sensitive nitrogen fixation mutants of Azotobacter vinelandii were obtained by nitrosoguanidine mutagenesis and penicillin selection. The mutants were unable to grow on N₂ at 39° but grew normally at 30° on N₂ and at both temperatures in the presence of metabolizable nitrogen compounds. Growth experiments and assays of whole cells for nitrogenase activity separated the mutants into two classes: 1. mutants in which the nitrogenase activity present in cells grown at 30° was unaffected by a shift to 39°, and 2. mutants which lost their nitrogen fixation activity after such a temperature shift. Assays of cell-free extracts of the second class of mutants showed that in all cases tested the enzymatic activity of the nitrogenase complex itself was not affected by the mutation. These mutants might therefore contain some other temperature-sensitive proteins specifically involved in nitrogen fixation.