Noncontact dipole effects on channel permeation. I. Experiments with (5F-indole)Trp13 gramicidin A channels.

Gramicidin A (gA), with four Trp residues per monomer, has an increased conductance compared to its Phe replacement analogs. When the dipole moment of the Trp13 side chain is increased by fluorination at indole position 5 (FgA), the conductance is expected to increase further. gA and FgA conductances to Na+, K+, and H+ were measured in planar diphytanoylphosphatidylcholine (DPhPC) or glycerylmonoolein (GMO) bilayers. In DPhPC bilayers, Na+ and K+ conductances increased upon fluorination, whereas in GMO they decreased. The low ratio in the monoglyceride bilayer was not reversed in GMO-ether bilayers, solvent-inflated or -deflated bilayers, or variable fatty acid chain monoglyceride bilayers. In both GMO and DPhPC bilayers, fluorination decreased conductance to H+ but increased conductance in the mixed solution, 1 M KCl at pH 2.0, where K+ dominates conduction. Eadie-Hofstee plot slopes suggest similar destabilization of K+ binding in both lipids. Channel lifetimes were not affected by fluorination in either lipid. These observations indicate that fluorination does not change the rotameric conformation of the side chain. The expected difference in the rate-limiting step for transport through channels in the two bilayers qualitatively explains all of the above trends.     

Tracing the Evolution of Competence in Haemophilus influenzae

Natural competence is the genetically encoded ability of some bacteria to take up DNA from the environment. Although most of the incoming DNA is degraded, occasionally intact homologous fragments can recombine with the chromosome, displacing one resident strand. This potential to use DNA as a source of both nutrients and genetic novelty has important implications for the ecology and evolution of competent bacteria. However, it is not known how frequently competence changes during evolution, or whether non-competent strains can persist for long periods of time. We have previously studied competence in H. influenzae and found that both the amount of DNA taken up and the amount recombined varies extensively between different strains. In addition, several strains are unable to become competent, suggesting that competence has been lost at least once. To investigate how many times competence has increased or decreased during the divergence of these strains, we inferred the evolutionary relationships of strains using the largest datasets currently available. However, despite the use of three datasets and multiple inference methods, few nodes were resolved with high support, perhaps due to extensive mixing by recombination. Tracing the evolution of competence in those clades that were well supported identified changes in DNA uptake and/or transformation in most strains. The recency of these events suggests that competence has changed frequently during evolution but the poor support of basal relationships precludes the determination of whether non-competent strains can persist for long periods of time. In some strains, changes in transformation have occurred that cannot be due to changes in DNA uptake, suggesting that selection can act on transformation independent of DNA uptake.     

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Use of C-banding for identifying radiation induced micronuclei

Differentiation of micronuclei (MN) caused by ionizing radiation from those caused by chemicals is a crucial step for managing treatment of individuals exposed to radiation. MN in binucleated lymphocytes in peripheral blood are widely used as biomarkers for estimating dose of radiation, but they are not specific for ionizing radiation. MN induced by ionizing radiation originate predominantly as a result of chromosome breaks (clastogenic action), whereas MN caused by chemical agents are derived from the loss of entire chromosomes (aneugenic action). C-banding highlights centromeres, which might make it possible to distinguish radiation induced MN, i.e., as a byproduct of acentric fragments, from those caused by the loss of entire chromosomes. To test the use of C-banding for identifying radiation induced MN, a blood sample from a healthy donor was irradiated with 3 Gy of Co-60 gamma rays and cultured. Cells were harvested and dropped onto slides, divided into a group stained directly with Giemsa and another processed for C banding, then stained with Giemsa. The frequency of MN in 500 binucleated cells was scored for each method. In preparations stained with Giemsa directly, the MN appeared as uniformly stained structures, whereas after C banding, some MN exhibited darker regions corresponding to centromeres that indicated that they were not derived from acentric fragments. The C-banding technique enables differentiation of MN from acentric chromosomal material. This distinction is useful for improving the specificity of the MN assay as a biomarker for ionizing radiation.     

Regulatory light chain phosphorylation and N-terminal extension increase cross-bridge binding and power output in Drosophila at in vivo myofilament lattice spacing

The N-terminal extension and phosphorylation of the myosin regulatory light chain (RLC) independently improve Drosophila melanogaster flight performance. Here we examine the functional and structural role of the RLC in chemically skinned fibers at various thick and thin filament lattice spacings from four transgenic Drosophila lines: rescued null or control (Dmlc2⁺), truncated N-terminal extension (Dmlc2^Δ2-46), disrupted myosin light chain kinase phosphorylation sites (Dmlc2^S66A,S67A), and dual mutant (Dmlc2^{Δ2-46; S66A,S67A}). The N-terminal extension truncation and phosphorylation sites disruption mutations decreased oscillatory power output and the frequency of maximum power output in maximally Ca²⁺-activated fibers compressed to near in vivo inter-thick filament spacing, with the phosphorylation sites disruption mutation having a larger affect. The diminished power output parameters with the N-terminal extension truncation and phosphorylation sites disruption mutations were due to the reduction of the number of strongly-bound cross-bridges and rate of myosin force production, with the larger parameter reductions in the phosphorylation sites disruption mutation additionally related to reduced myosin attachment time. The phosphorylation and N-terminal extension-dependent boost in cross-bridge kinetics corroborates previous structural data, which indicate these RLC attributes play a complementary role in moving and orienting myosin heads toward actin target sites, thereby increasing fiber and whole fly power generation.     

Effects of headwater impoundment and channelization on invertebrate drift

The construction of a flood control impoundment on Twitty's Creek added large numbers of organisms of limnetic origin to the stream ecosystem. However, the number of limnetic organisms per unit volume of water decreased rapidly as the distance downstream from the reservoir increased and, during most sampling periods, made up an insignificant portion of the total drift biomass at 7.2 km downstream. Factors favoring the extended downstream drift of limnetic organisms were high stream discharge and low water temperature.Several taxa of benthic organisms had much lower drift rates in the station immediately below the dam than at other stations and several taxa commonly taken at other stations were not captured immediately below the reservoir outfall. One possible explanation is that these organisms may have longer drift recruitment distances than the distance from the reservoir outfall to the sample location.A comparison of drift densities of organisms of benthic origin and benthic standing crop densities in channeled and unchanneled streams revealed that drift densities were higher in channeled streams than in unchanneled streams for most taxa of invertebrates. In addition, channeled streams appeared to have lower benthic standing crops than unchanneled streams for most taxa of invertebrates.In stream sections impacted by either channelization or the Twitty Lake outfall, the energy dynamics of the stream ecosystems were altered by increased density of drifting invertebrates. From the standpoint of increasing food availability to the fish fauna of the stream, these changes would appear to benefit drift feeding species and negatively impact bottom feeding species.