The synthesis of 15N- and deuterium-substituted, spin-labeled analogues of NAD+ and their use in EPR studies of dehydrogenases

Calcium efflux and EGTA-induced calcium release from an internal platelet membrane fraction have been studied after the oxalate-supported calcium uptake had reached steady state. Increasing external calcium concentrations stimulate the calcium efflux velocity, with an apparent half-maximal stimulation at about 5 microM outside calcium concentration and a maximal velocity of calcium efflux of 4.66 +/- 2.32 nmol X min-1 X mg-1. Moreover, the ratio of the liberated calcium on the loaded calcium seems to be independent of the increasing external calcium concentration. Increasing the calculated internal calcium concentration by varying the oxalate potassium concentration from 10 mM to 1 mM results in an increase of the liberated calcium from the membrane vesicles from 7.4% to 63%, respectively, without changing the calcium efflux velocity. Similar conclusions can be drawn from the observation of results from the calcium efflux and EGTA-induced calcium release methods. Moreover, calcium pump reversal does not seem to be responsible for the calcium efflux or calcium release. All these different points added to the previously described regulation of calcium efflux by the catalytic subunit of cAMP protein kinase suggest us that the mechanism of calcium liberation by the platelet membranes is different from the calcium uptake.     

The cDNA cloning and immunological characterization of hamster p53.

We have cloned and sequenced the p53-encoding cDNA of Syrian hamster. The encoded product is 78% and 75% homologous to human and mouse p53, respectively. Immunoprecipitations of the cDNA-encoded protein by monoclonal antibodies specific for mammalian p53 confirmed the identity of the protein.     

Assessing Greater Sage-Grouse Selection of Brood-Rearing Habitat Using Remotely-Sensed Imagery: Can Readily Available High-Resolution Imagery Be Used to Identify Brood-Rearing Habitat Across a Broad Landscape?

Greater sage-grouse populations have decreased steadily since European settlement in western North America. Reduced availability of brood-rearing habitat has been identified as a limiting factor for many populations. We used radio-telemetry to acquire locations of sage-grouse broods from 1998 to 2012 in Strawberry Valley, Utah. Using these locations and remotely-sensed NAIP (National Agricultural Imagery Program) imagery, we 1) determined which characteristics of brood-rearing habitat could be used in widely available, high resolution imagery 2) assessed the spatial extent at which sage-grouse selected brood-rearing habitat, and 3) created a predictive habitat model to identify areas of preferred brood-rearing habitat. We used AIC model selection to evaluate support for a list of variables derived from remotely-sensed imagery. We examined the relationship of these explanatory variables at three spatial extents (45, 200, and 795 meter radii). Our top model included 10 variables (percent shrub, percent grass, percent tree, percent paved road, percent riparian, meters of sage/tree edge, meters of riparian/tree edge, distance to tree, distance to transmission lines, and distance to permanent structures). Variables from each spatial extent were represented in our top model with the majority being associated with the larger (795 meter) spatial extent. When applied to our study area, our top model predicted 75% of naïve brood locations suggesting reasonable success using this method and widely available NAIP imagery. We encourage application of our methodology to other sage-grouse populations and species of conservation concern.     

Construction of first-generation lactococcal integrative cloning vectors

Using a randomly-cloned, HindIII-digested, chromosomal fragment from Lactococcus lactis subsp. lactis LM0230, first-generation lactococcal integrative cloning vectors were developed. Through dideoxy DNA sequence analysis, the cloned chromosomal DNA fragment was determined to be 1026 base pairs. Southern hybridization studies demonstrated applicability of the integrative vector to other strains of L. lactis and L. lactis subsp. cremoris. Identification of a single NruI site near the middle of the chromosomal fragment allowed insertion of the erythromycin (Em)-resistance (ery ^r) gene obtained from L. lactis IL1837. Integration of the ery ^r gene into the L. lactis LM0230 chromosome was achieved by a Campbell-like recombination. The nisin (Nis)-resistance (nis ^r) gene from L. lactis IL1904 was inserted into the NruI site in a separate clone and integration into the L. lactis LM0230 chromosome was achieved via a replacement recombination event following electroporation of the linearized nis ^r fragment flanked by the cloned chromosomal DNA. Transformants grown in the absence of either Em or Nis for >200 generations and subsequently transferred to various concentrations of the selectable agent confirmed the stability of the integrated genes. Further studies involving the Nis-resistant (Nis ^r ) transformant suggested that the integrated nis ^r gene may be amplifying within the host chromosome. Correspondence to: S. K. Harlander     

Dynamic resource allocation between pre- and postcopulatory episodes of sexual selection determines competitive fertilization success

In polyandrous mating systems, male reproductive success depends on both mate-acquisition traits (precopulatory) and sperm competitive abilities (postcopulatory). Empirical data on the interaction between these traits are inconsistent; revealing positive, negative or no relationships. It is generally expected that the investment in pre- and postcopulatory traits is mediated by environmental conditions. To test how dietary resource availability affects sexual ornamentation, sperm quality and their interrelationship in three-spined sticklebacks (Gasterosteus aculeatus), full-sibling groups were raised under three conditions differing in food quantity and/or quality (i.e. carotenoid content): (i) high-quantity/high-quality, (ii) high-quantity/low-quality or (iii) low-quantity/low-quality. After 1 year of feeding, food-restricted males developed a more intense breeding coloration and faster sperm compared with their well-fed brothers, indicating that they allocated relatively more in pre- and postcopulatory traits. Moreover, they outcompeted their well-fed, carotenoid-supplemented brothers in sperm competition trials with equal numbers of competing sperm, suggesting that food-restricted males maximize their present reproductive success. This may result in reduced future reproductive opportunities as food-restricted males suffered from a higher mortality, had an overall reduced body size, and sperm number available for fertilization. In accordance with theory, a trade-off between the investment in pre- and postcopulatory traits was observed in food-restricted males, whereas well-fed males were able to allocate to both traits resulting in a significantly positive relationship.     

The role of aromatic side chain residues in micelle binding by pancreatic colipase. Fluorescence studies of the porcine and equine proteins.

Fluorescence techniques have been employed to study the interaction of porcine and equine colipase with pure taurodeoxycholate and mixed micelles. Nitrotyrosine-55 of porcine colipase is obtained by modification with tetranitromethane (low excess, in the presence of taurodeoxycholate) of the protein followed by gel filtration and ion-exchange chromatography. Verification of the residue modified was obtained by h.p.l.c. peptide purification and sequence analysis. Reduction and quantitative reaction with dansyl chloride yields a fluorescent derivative that is twice as active in conjunction with lipase as is native colipase and that exhibits a strong emission band at 550 nm. Addition of micellar concentrations of taurodeoxycholate causes a 4.3-fold increase in the emission maximum as well as a 70 nm blue shift to 480 nm. Inclusion of oleic acid to form a mixed micelle reduces these spectral effects. Scatchard analysis of the data yield a Kd of 6.8 X 10(-4) M and a single colipase-binding site for taurodeoxycholate micelles. The data, by analogy to a phospholipase system, are consistent with a direct insertion of dansyl-NH-tyrosine-55 into the micelle. The presence of a single tryptophan residue (Trp-52) in equine colipase provides an intrinsic fluorescent probe for studying protein-micelle interaction. The emission maximum of horse colipase at 345 nm indicates a solvent-accessible tryptophan residue which becomes less so on binding of micelles. A blue shift of 8 nm and a 2-fold increase in amplitude is indicative of a more hydrophobic environment for tryptophan induced by taurodeoxycholate micelles. There is also a decrease in KSV for acrylamide quenching in the presence of micelles, which further supports a loss of solvent accessibility. The most dramatic pH effects are observed with KI quenching, and may indicate the presence of negative charges near Trp-52.