14-3-3 proteins double the number of outward-rectifying K+ channels available for activation in tomato cells

Outward-rectifying K+ channels are modulated in response to environmental stimuli by a range of intracellular factors, such as cytoplasmic Ca2+, pH, kinases and phosphatases. Here we report that voltage-dependent outward-rectifying K+ channels in tomato cells are also targets for modulation by 14-3-3 proteins. In whole-cell patch-clamp experiments, recombinant 14-3-3 protein (tomato isoform TFT7) was introduced into tomato cell protoplasts via the patch pipette. As a result the steady-state outward K+ current increased twofold and this increase was not dependent upon the presence of cytoplasmic ATP. A phosphorylated peptide that contained a phosphorylated 14-3-3 target-binding motif (RSTS*TP), derived from nitrate reductase, blocked the effect of 14-3-3, thus showing the specific nature of 14-3-3 action. Kinetic parameters of the conductance, like (de)activation kinetics, voltage dependence of gating and activation potential, were not significantly different between control and 14-3-3 infused cells. Analysis of single-channel activity and whole-cell noise indicated that the single-channel conductance was not affected by 14-3-3 infusion. We conclude that 14-3-3 proteins recruit 'sleepy' channels into a voltage-activatable state. The molecular mechanism underlying the 1 : 1 ratio of constitutively active and 14-3-3 recruited channels is discussed in the light of known functions of 14-3-3 dimers.     

DNA evolutionary rates in nine-primaried passerine birds

Differences in single-copy nuclear-DNA sequences among 13 species of passerine birds were measured using DNA-DNA hybridization. A matrix of pairwise dissimilarity values (delta mode distances) was constructed from analysis of fitted thermal dissociation curves. A least-squares method of phylogenetic estimation was used to construct two topologies from the distance matrix, one constraining branch lengths of sister taxa to be equal and the other permitting such lengths to vary. These topologies were identical in the pattern of branching of taxa, and the difference in their sums of squares was not statistically significant, suggesting that rates of DNA evolution in sister groups of nine- primaried oscines are equal. A nonparametric test for nonrandom variation in distances of sister groups to outgroup taxa revealed no statistically significant deviation from random variation that would be expected as a result of measurement error. However, the level of measurement error was such that rates of DNA evolution in sister taxa could vary by as much as 10% without being detected with the statistical methods used here.      

Enumeration of nitrite-oxidizing bacteria in grassland soils using a Most Probable Number technique: Effect of nitrite concentration and sampling procedure

Abstract Numbers of nitrite-oxidizing bacteria were determined in grassland soils using a Most Probable Number technique. Two concentrations of nitrite were used in the incubation medium, i.e. 0.05 and 5.0 mM. The results of the enumerations were highly dependent on the nitrite concentration as well as on the grassland soil sampled. In the one soil the highest numbers were counted with 5.0 mM, whereas in other soils highest numbers were obtained in media with 0.05 mM nitrite. The spatial distribution of nitrite-oxidizing bacteria was determined i two field plots. Variation between individual samples was low in one plot and high in the other. The implications of the observed differences for the sampling procedure in each sampling plot are discussed. In the waterlogged, oxygen-limited soils, numbers of nitrite-oxidizing bacteria were as high as in the drained soils. The chemolitho-autotrophic nature of these nitrifiers has been confirmed.     

ACUTE EFFECTS OF MOVEMENT VELOCITY ON BLOOD LACTATE AND GROWTH HORMONE RESPONSES AFTER ECCENTRIC BENCH PRESS EXERCISE IN RESISTANCE-TRAINED MEN

This study aimed to compare the effects of different velocities of eccentric muscle actions on acute blood lactate and serum growth hormone (GH) concentrations following free weight bench press exercises performed by resistance-trained men. Sixteen healthy men were divided into two groups: slow eccentric velocity (SEV; n = 8) and fast eccentric velocity (FEV; n = 8). Both groups performed four sets of eight eccentric repetitions at an intensity of 70% of their one repetition maximum eccentric (1RMecc) test, with 2-minute rest intervals between sets. The eccentric velocity was controlled to 3 seconds per range of motion for SEV and 0.5 seconds for the FEV group. There was a significant difference (P < 0.001) in the kinetics of blood lactate removal (at 3, 6, 9, 15, and 20 min) and higher mean values for peak blood lactate (P = 0.001) for the SEV group (9.1 ± 0.5 mM) compared to the FEV group (6.1 ± 0.4 mM). Additionally, serum GH concentrations were significantly higher (P < 0.001) at 15 minutes after bench press exercise in the SEV group (1.7 ± 0.6 ng · mL⁻¹) relative to the FEV group (0.1 ± 0.0 ng · mL⁻¹). In conclusion, the velocity of eccentric muscle action influences acute responses following bench press exercises performed by resistance-trained men using a slow velocity resulting in a greater metabolic stress and hormone response.     

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

Background

Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods.

Results

Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group.

Conclusion

The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice.     

Genomic flank-sequencing of plasposon insertion sites for rapid identification of functional genes

Plasposons are modified mini-Tn5 transposons for random mutagenesis of Gram-negative bacteria. Their unique design allows for the rescue cloning and sequencing of DNA that flanks insertion sites in plasposon mutants. However, this process can be laborious and time-consuming, as it involves genomic DNA isolation, restriction endonuclease treatment, subsequent religation, transformation of religated DNA into an Escherichia coli host, and re-isolation as a plasmid, which is then used as a template in sequencing reactions with primers that read from the plasposon ends into the flanking DNA regions. We describe here a method that produces flanking DNA sequences directly from genomic DNA that is isolated from plasposon mutants. By eliminating the need for rescue cloning, our protocol dramatically reduces time and effort, typically by 2 to 3 working days, as well as costs associated with digestion, ligation, transformation, and plasmid isolation. Furthermore, it allows for a high-throughput automated approach to analysis of the plasposome, i.e. the collective set of plasposon insertion sites in a plasposon mutant library. We have tested the utility of genomic flank-sequencing on three plasposon mutants of the soil bacterium Collimonas fungivorans with abolished ability to degrade chitin.     

Most Probable Numbers of chemolitho-autotrophic nitrite-oxidizing bacteria in well drained grassland soils: Stimulation by high nitrite concentrations

Abstract The results of Most Probable Number (MPN) enumerations of chemolitho-autotrophic nitrite oxidizers are very much dependent on the nitrite concentration applied in the incubation medium. In order to explain this dependency, the influence of pH, nitrite and resultant nitrous acid concentration of the incubation medium on the MPN-enumeration was investigated. It appeared that none of these factors were exclusively responsible for the result of the enumeration. In samples from a well drained grassland soil, highest number have been obtained with a combination of a low pH and a low nitrite concentration in the counting medium. The relation between the MPN-counting results and the nitrous acid concentration showed an optimum with the same soil samples. It was hypothesized that the relatively high numbers of nitrite-oxidizing cells determined in soil samples at a high nitrite concentration and pH 7.3 was due to the presence of dormant cells. However, this hypothesis could not be confirmed with enumerations of aged cell suspensions of different Nitrobacter species. In contrast to the field observations, these resting cells were always enumerated more efficiently at a low nitrite concentration. The importance of the use of more than one incubation medium for the enumeration of nitrite-oxidizing bacteria is emphasized.