Too many mutants with multiple mutations

It has recently become clear that the classical notion of the random nature of mutation does not hold for the distribution of mutations among genes: most collections of mutants contain more isolates with two or more mutations than predicted by the mutant frequency on the assumption of a random distribution of mutations. Excesses of multiples are seen in a wide range of organisms, including riboviruses, DNA viruses, prokaryotes, yeasts, and higher eukaryotic cell lines and tissues. In addition, such excesses are produced by DNA polymerases in vitro. These "multiples" appear to be generated by transient, localized hypermutation rather than by heritable mutator mutations. The components of multiples are sometimes scattered at random and sometimes display an excess of smaller distances between mutations. As yet, almost nothing is known about the mechanisms that generate multiples, but such mutations have the capacity to accelerate those evolutionary pathways that require multiple mutations where the individual mutations are neutral or deleterious. Examples that impinge on human health may include carcinogenesis and the adaptation of microbial pathogens as they move between individual hosts.     

Bilirubin as an antioxidant in micelles and lipid bilayers: its contribution to the total antioxidant capacity of human blood plasma

The antioxidant capacities, antioxidant activities, k(inh), and stoichiometric factors, n, of water-soluble derivatives of bilirubin (BR), BR-human serum albumin (BR-HSA), and BR-ditaurate disodium conjugate (BRC) were determined in aqueous/lipid dispersions of sodium dodecyl sulfate (SDS) micelles/methyl linoleate and in bilayers of dilinoleoylphosphatidylcholine (DLPC) during initiation by water-soluble azo-bis-amidinopropane dihydrochloride (ABAP). The inhibition rate constants for BRC and BR-HSA were similar in micelles (k(inh) approximately 1.3 x 10(4) M(-1) s(-1)), where n approximately 2, whereas the k(inh) for BR-HSA dropped by (1/2) in bilayers. The dimethyl ester of bilirubin (BRDE) gave a k(inh) only one-tenth that of the vitamin E analog, pentamethylhydroxychroman (PMHC) in SDS micelles/methyl linoleate when initiated by lipid-soluble azo-bis-2,4-dimethylvaleronitrile (DMVN). Biliverdin hydrochloride (BVHCl) was NOT an effective peroxyl radical-trapping agent in the micellar phase during initiation by ABAP or DMVN containing methyl linoleate but it inhibited oxygen uptake in the aqueous phase. Both BRC and BR-HSA extended the total radical antioxidant parameter (TRAP) of human blood plasma and their contribution to TRAP was in the range of 5-10% of the natural TRAP of blood plasma, depending on the BR content determined in the blood plasma.     

Vegetation classification,mapping, and monitoring at Voyageurs National Park,Minnesota: An application of the U.S. National Vegetation Classification

Question: How can the U.S. National Vegetation Classification (USNVC) serve as an effective tool for classifying and mapping vegetation, and inform assessments and monitoring? Location: Voyageurs National Park, northern Minnesota, U.S.A and environs. The park contains 54 243 ha of terrestrial habitat in the sub-boreal region of North America. Methods: We classified and mapped the natural vegetation using the USNVC, with ‘alliance’and ‘association’as base units. We compiled 259 classification plots and 1251 accuracy assessment test plots. Both plot and type ordinations were used to analyse vegetation and environmental patterns. Color infrared aerial photography (1:15840 scale) was used for mapping. Polygons were manually drawn, then transferred into digital form. Classification and mapping products are stored in publicly available databases. Past fire and logging events were used to assess distribution of forest types. Results and Discussion: Ordination and cluster analyses confirmed 49 associations and 42 alliances, with three associations ranked as globally vulnerable to extirpation. Ordination provided a useful summary of vegetation and ecological gradients. Overall map accuracy was 82.4%. Pinus banksiana - Picea mariana forests were less frequent in areas unburned since the 1930s. Conclusion: The USNVC provides a consistent ecological tool for summarizing and mapping vegetation. The products provide a baseline for assessing forests and wetlands, including fire management. The standardized classification and map units provide local to continental perspectives on park resources through linkages to state, provincial, and national classifications in the U.S. and Canada, and to NatureServe's Ecological Systems classification.     

Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion

To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.     

Structure and orientation of a G protein fragment in the receptor bound state from residual dipolar couplings

Residual dipolar couplings for a ligand that is in fast exchange between a free state and a state where it is bound to a macroscopically ordered membrane protein carry precise information on the structure and orientation of the bound ligand. The couplings originate in the bound state but can be detected on the free ligand using standard high resolution NMR. This approach is used to study an analog of the C-terminal undecapeptide of the alpha-subunit of the heterotrimeric G protein transducin when bound to photo-activated rhodopsin. Rhodopsin is the major constituent of disk-shaped membrane vesicles from rod outer segments of bovine retinas, which align spontaneously in the NMR magnet. Photo-activation of rhodopsin triggers transient binding of the peptide, resulting in measurable dipolar contributions to 1J(NH) and 1J(CH) splittings. These dipolar couplings report on the time-averaged orientation of bond vectors in the bound peptide relative to the magnetic field, i.e. relative to the membrane normal. Approximate distance restraints of the bound conformation were derived from transferred NOEs, as measured from the difference of NOESY spectra recorded prior to and after photo-activation. The N-terminal eight residues of the bound undecapeptide adopt a near-ideal alpha-helical conformation. The helix is terminated by an alpha(L) type C-cap, with Gly9 at the C' position in the center of the reverse turn. The angle between the helix axis and the membrane normal is 40 degrees (+/-4) degrees. Peptide protons that make close contact with the receptor are identified by analysis of the NOESY cross-relaxation pattern and include the hydrophobic C terminus of the peptide.     

Propagule pressure and persistence in experimental populations

Average inoculum size and number of introductions are known to have positive effects on population persistence. However, whether these factors affect persistence independently or interact is unknown. We conducted a two-factor experiment in which 112 populations of parthenogenetic Daphnia magna were maintained for 41 days to study effects of inoculum size and introduction frequency on: (i) population growth, (ii) population persistence and (iii) time-to-extinction. We found that the interaction of inoculum size and introduction frequency-the immigration rate-affected all three dependent variables, while population growth was additionally affected by introduction frequency. We conclude that for this system the most important aspect of propagule pressure is immigration rate, with relatively minor additional effects of introduction frequency and negligible effects of inoculum size.