Asymmetric somatic hybridization between tall fescue (Festuca arundinacea Schreb.) and irradiated Italian ryegrass (Lolium multiflorum Lam.) protoplasts

Intergeneric asymmetric somatic hybrids have been obtained by the fusion of metabolically inactivated protoplasts from embryogenic suspension cultures ofFestuca arundinacea (recipient) and protoplasts from a non-morphogenic cell suspension ofLolium multiflorum (donor) irradiated with 10, 25, 50, 100, 250 and 500 Gy of X-rays. Regenerating calli led to the recovery of genotypically and phenotypically different asymmetric somatic hybridFestulolium plants. The genome composition of the asymmetric somatic hybrid clones was characterized by quantitative dot-blot hybridizations using dispersed repetitive DNA sequences specific to tall fescue and Italian ryegrass. Data from dot-blot hybridizations using two cloned Italian ryegrass-specific sequences as probes showed that irradiation favoured a unidirectional elimination of most or part of the donor chromosomes in asymmetric somatic hybrid clones obtained from fusion experiments using donor protoplasts irradiated at doses 250 Gy. Irradiation of cells of the donor parent with 500 Gy prior to protoplast fusion produced highly asymmetric nuclear hybrids with over 80% elimination of the donor genome as well as clones showing a complete loss of donor chromosomes. Further information on the degree of asymmetry in regenerated hybrid plants was obtained from chromosomal analysis including in situ hybridizations withL. multiflorum-specific repetitive sequences. A Southern blot hybridization analysis using one chloroplast and six mitochondrial-specific probes revealed preferentially recipient-type organelles in asymmetric somatic hybrid clones obtained from fusion experiments with donor protoplasts irradiated with doses higher than 100 Gy. It is concluded that the irradiation of donor cells before fusion at different doses can be used for producing both nuclear hybrids with limited donor DNA elimination or highly asymmetric nuclear hybrid plants in an intergeneric graminaceous combination. For a wide range of radiation doses tested (25–250Gy), the degree of the species-specific genome elimination from the irradiated partner seems not to be dose dependent. A bias towards recipient-type organelles was apparent when extensive donor nuclear genome elimination occurred.Abbreviations cpDNA Chloroplast DNA - 2, 4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - IOA iodoacetamide - mtDNA mitochondrial DNA - RFLP restriction fragment length polymorphism     

Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes

Purified (Na+ + K+)-ATPase from pig kidney was attached to black lipid membranes and ATP-induced electric currents were measured as described previously by Fendler et al. ((1985) EMBO J. 4, 3079-3085). An ATP concentration jump was produced by an ultraviolet-light flash converting non-hydrolysable caged ATP to ATP. In the presence of Na+ and Mg2+ this resulted in a transient current signal. The pump current was not only ATP dependent, but also was influenced by the ATP/caged ATP ratio. It was concluded that caged ATP binds to the enzyme (and hence inhibits the signal) with a Ki of approx. 30 microM, which was confirmed by enzymatic activity studies. An ATP affinity of approx. 2 microM was determined. The addition of the protonophore 1799 and the Me+/H+ exchanger monensin made the bilayer conductive leading to a stationary pump current. The stationary current was strongly increased by the addition of K+ with a K0.5 of 700 microM. Even in the absence of K+ a stationary current could be measured, which showed two Na+-affinities: a high-affinity (K0.5 less than or equal to 1 mM) and a low-affinity (K0.5 greater than or equal to 0.2 M). In order to explain the sustained electrogenic Na+ transport during the Na+-ATPase activity, it is proposed, that Na+ can replace K+ in dephosphorylating the enzyme, but binds about 1000-times weaker than K+. The ATP requirement of the Na+-ATPase was the same (K0.5 = 2 microM) with regard to the peak currents and the stationary currents. However, for the (Na+ + K+)-ATPase the stationary currents required more ATP. The results are discussed on the basis of the Albers-Post scheme.     

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner. The amount of sample and number of steps is minimized and no biochemical reformulation is required. In the current application a robotic liquid handling system enables high-throughput use, but the technique can easily be adapted in a nonautomated setting. This method has been applied successfully for the rapid optimization of crystallization conditions in nine representative cases.     

Quantitative comparison of DNA detection by GFP-lac repressor tagging,fluorescence in situ hybridization and immunostaining

Background  

GFP-fusion proteins and immunostaining are methods broadly applied to investigate the three-dimensional organization of cells and cell nuclei, the latter often studied in addition by fluorescence in situ hybridization (FISH). Direct comparisons of these detection methods are scarce, however.     

The cold‐water climate shield: delineating refugia for preserving salmonid fishes through the 21st century

The distribution and future fate of ectothermic organisms in a warming world will be dictated by thermalscapes across landscapes. That is particularly true for stream fishes and cold‐water species like trout, salmon, and char that are already constrained to high elevations and latitudes. The extreme climates in those environments also preclude invasions by most non‐native species, so identifying especially cold habitats capable of absorbing future climate change while still supporting native populations would highlight important refugia. By coupling crowd‐sourced biological datasets with high‐resolution stream temperature scenarios, we delineate network refugia across >250 000 stream km in the Northern Rocky Mountains for two native salmonids—bull trout (BT) and cutthroat trout (CT). Under both moderate and extreme climate change scenarios, refugia with high probabilities of trout population occupancy (>0.9) were predicted to exist (33–68 BT refugia; 917–1425 CT refugia). Most refugia are on public lands (>90%) where few currently have protected status in National Parks or Wilderness Areas (<15%). Forecasts of refuge locations could enable protection of key watersheds and provide a foundation for climate smart planning of conservation networks. Using cold water as a ‘climate shield’ is generalizable to other species and geographic areas because it has a strong physiological basis, relies on nationally available geospatial data, and mines existing biological datasets. Importantly, the approach creates a framework to integrate data contributed by many individuals and resource agencies, and a process that strengthens the collaborative and social networks needed to preserve many cold‐water fish populations through the 21st century.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

Über die Wirkung verschiedener Faktoren,insbesondere narkotisierender Substanzen auf die vitale Methylenblaufärbung bei in vitro gezüchteten Fibrocyten

Ohne Zusammenfassung