Spatial and temporal genetic structure of a river‐resident Atlantic salmon (Salmo salar) after millennia of isolation

The river‐resident Salmo salar (“småblank”) has been isolated from other Atlantic salmon populations for 9,500 years in upper River Namsen, Norway. This is the only European Atlantic salmon population accomplishing its entire life cycle in a river. Hydropower development during the last six decades has introduced movement barriers and changed more than 50% of the river habitat to lentic conditions. Based on microsatellites and SNPs, genetic variation within småblank was only about 50% of that in the anadromous Atlantic salmon within the same river. The genetic differentiation (F_ST) between småblank and the anadromous population was 0.24. This is similar to the differentiation between anadromous Atlantic salmon in Europe and North America. Microsatellite analyses identified three genetic subpopulations within småblank, each with an effective population size N_e of a few hundred individuals. There was no evidence of reduced heterozygosity and allelic richness in contemporary samples (2005–2008) compared with historical samples (1955–56 and 1978–79). However, there was a reduction in genetic differentiation between sampling localities over time. SNP data supported the differentiation of småblank into subpopulations and revealed downstream asymmetric gene flow between subpopulations. In spite of this, genetic variation was not higher in the lower than in the upper areas. The meta‐population structure of småblank probably maintains genetic variation better than one panmictic population would do, as long as gene flow among subpopulations is maintained. Småblank is a unique endemic island population of Atlantic salmon. It is in a precarious situation due to a variety of anthropogenic impacts on its restricted habitat area. Thus, maintaining population size and avoiding further habitat fragmentation are important.     

Concentration dependence of alpha-synuclein fibril length assessed by quantitative atomic force microscopy and statistical-mechanical theory

The initial concentration of monomeric amyloidogenic proteins is a crucial factor in the in vitro formation of amyloid fibrils. We use quantitative atomic force microscopy to study the effect of the initial concentration of human α-synuclein on the mean length of mature α-synuclein fibrils, which are associated with Parkinson's disease. We determine that the critical initial concentration, below which low-molecular-weight species dominate and above which fibrils are the dominant species, lies at ∼15 μM, in good agreement with earlier measurements using biochemical methods. In the concentration regime where fibrils dominate, we find that their mean length increases with initial concentration. These results correspond well to the qualitative predictions of a recent statistical-mechanical model of amyloid fibril formation. In addition, good quantitative agreement of the statistical-mechanical model with the measured mean fibril length as a function of initial protein concentration, as well as with the fibril length distributions for several protein concentrations, is found for reasonable values of the relevant model parameters. The comparison between theory and experiment yields, for the first time to our knowledge, an estimate of the magnitude of the free energies associated with the intermolecular interactions that govern α-synuclein fibril formation.     

Low-Intensity Agricultural Landscapes in Transylvania Support High Butterfly Diversity: Implications for Conservation

European farmland biodiversity is declining due to land use changes towards agricultural intensification or abandonment. Some Eastern European farming systems have sustained traditional forms of use, resulting in high levels of biodiversity. However, global markets and international policies now imply rapid and major changes to these systems. To effectively protect farmland biodiversity, understanding landscape features which underpin species diversity is crucial. Focusing on butterflies, we addressed this question for a cultural-historic landscape in Southern Transylvania, Romania. Following a natural experiment, we randomly selected 120 survey sites in farmland, 60 each in grassland and arable land. We surveyed butterfly species richness and abundance by walking transects with four repeats in summer 2012. We analysed species composition using Detrended Correspondence Analysis. We modelled species richness, richness of functional groups, and abundance of selected species in response to topography, woody vegetation cover and heterogeneity at three spatial scales, using generalised linear mixed effects models. Species composition widely overlapped in grassland and arable land. Composition changed along gradients of heterogeneity at local and context scales, and of woody vegetation cover at context and landscape scales. The effect of local heterogeneity on species richness was positive in arable land, but negative in grassland. Plant species richness, and structural and topographic conditions at multiple scales explained species richness, richness of functional groups and species abundances. Our study revealed high conservation value of both grassland and arable land in low-intensity Eastern European farmland. Besides grassland, also heterogeneous arable land provides important habitat for butterflies. While butterfly diversity in arable land benefits from heterogeneity by small-scale structures, grasslands should be protected from fragmentation to provide sufficiently large areas for butterflies. These findings have important implications for EU agricultural and conservation policy. Most importantly, conservation management needs to consider entire landscapes, and implement appropriate measures at multiple spatial scales.     

In vitro regeneration and cytological characterization of shoots from leaf explants of three accessions of Solanum commersonii

In vitro culture of explants were used to apply genetic or cell engineering techniques to the sexually incompatible potato relative Solanum commersonii (2n=2x=24) Three accessions of S. commersonii were tested for regeneration from leaf explants using six different protocols. A two step-regeneration procedure gave the best results. Genetic variability for regeneration ability was found between accessions, and between clones within accessions. The accession PI 472834 regenerated at highest frequency. Clones with high regeneration ability were selected. Approximately 60% of regenerated plants were diploids and 40% were tetraploids. A very low frequency of chimeras was found. Leaf shape and chloroplast counts in guard cells were shown to be quick and reliable methods for estimating ploidy levels. Use of the diploid and tetraploid regenerants obtained for potato breeding is discussed.Abbreviations BAP 6-benzylaminopurine - EBN Endosperm Balance Number - GA₃ gibberellic acid - IAA indole-3-acetic acid - MS Murashige and Skoog - NAA 1-naphthalene-acetic acid - ZEA zeatin     

Targeting of large liposomes with lectins increases their binding to plant protoplasts

Soybean agglutinin, peanut agglutinin, and concanavalin A were covalently bound by condensation reaction to gangliosides and ceramides incorporated within the bilayer of multilamellar and unilamellar liposomes. These modified liposomes had a much higher affinity for carrot and tobacco protoplasts except when concanavalin A was used.

In addition, soybean agglutinin and concanavalin A were attached by ligand-specific binding to liposomes containing cholesterol molecules derivatized with each lectin-specific sugar. This procedure allowed efficient crosslinking of liposomes to protoplasts. The same effect was achieved with soybean agglutinin and peanut agglutinin when derivatized cholesterol was replaced by gangliosides. The implications of these findings for the liposome-mediated nucleic acid transfer into protoplasts are discussed.

     

Effects of chloramphenicol on plant cells: Potential as a selectable marker for transformation studies

The growth of tobacco and carrot cells in suspension cultures and on solidified medium was strongly inhibited by the antibiotic chloramphenicol. This effect was irreversible. The drug had no effect on [³H]-uridine incorporation but inhibited protein synthesis and induced striking morphological alterations in intoxicated cells. O₂ consumption was also reduced in treated cells. Tobacco and carrot cells were unable to inactivate chloramphenicol by enzymatic acetylation.     

Organization and sequence analysis of the 2,4-dichlorophenol hydroxylase and dichlorocatechol oxidative operons of plasmid pJP4.

Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorphenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro- and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and tfdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and clcD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.     

Enrichment for enhanced competitive plant root tip colonizers selects for a new class of biocontrol bacteria

Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl (Fusarium oxysporum f.sp. radicis-lycopersici ). Several bacteria have been described to be able to control the disease, using different mechanisms. Here we describe a method that enables us to select, after application of a crude rhizobacterial mixture on a sterile seedling, those strains that reach the root tip faster than our best tomato root colonizer tested so far, the Pseudomonas fluorescens biocontrol strain WCS365. Of the five tested new isolates, four appeared to be able to reduce the number of diseased plants. Analysis of one of these strains, P. fluorescens PCL1751, suggests that it controls the disease through the mechanism 'competition for nutrients and niches', a mechanism novel for biocontrol bacteria. Moreover, this is the first report describing a method to enrich for biocontrol strains from a crude mixture of rhizobacteria. Another advantage of the method is that four out of five strains do not produce antifungal metabolites, which is preferential for registration as a commercial product.     

Heat shock protein 27 is the major differentially phosphorylated protein involved in renal epithelial cellular stress response and controls focal adhesion organization and apoptosis

We used two-dimensional difference gel electrophoresis to determine early changes in the stress-response pathways that precede focal adhesion disorganization linked to the onset of apoptosis of renal epithelial cells. Treatment of LLC-PK1 cells with the model nephrotoxicant 1,2-(dichlorovinyl)-L-cysteine (DCVC) resulted in a >1.5-fold up- and down-regulation of 14 and 9 proteins, respectively, preceding the onset of apoptosis. Proteins included those involved in metabolism, i.e. aconitase and pyruvate dehydrogenase, and those related to stress responses and cytoskeletal reorganization, i.e. cofilin, Hsp27, and alpha-b-crystallin. The most prominent changes were found for Hsp27, which was related to a pI shift in association with an altered phosphorylation status of serine residue 82. Although both p38 and JNK were activated by DCVC, only inhibition of p38 with SB203580 reduced Hsp27 phosphorylation, which was associated with accelerated reorganization of focal adhesions, cell detachment, and apoptosis. In contrast, inhibition of JNK with SP600125 maintained cell adhesion as well as protection against apoptosis. Active JNK co-localized at focal adhesions after DCVC treatment in a FAK-dependent manner. Inhibition of active JNK localization at focal adhesions did not prevent DCVC-induced phosphorylation of Hsp27. Overexpression of a phosphorylation-defective mutant Hsp27 acted as a dominant negative and accelerated the DCVC-induced changes in the focal adhesions as well as the onset of apoptosis. Our data fit a model whereby early p38 activation results in a rapid phosphorylation of Hsp27, a requirement for proper maintenance of cell adhesion, thus suppressing renal epithelial cell apoptosis.