The cytotoxic effects of single-stranded telomere mimics on OMA-BL1 cells.

Telomerase is a ribonucleoprotein that adds 5'-d(TTAGGG)-3' hexameric repeats onto the 3' ends of chromosomes. High telomerase activity has been associated with immortal cells, transformed cells, mitogenic stimulation, and proliferative diseases. It is not clear what phenotype would be observed by transient inhibition of telomerase. Studies were designed to inhibit telomerase activity using a series of S-ODN telomere sequence motifs. The studies evaluated the length, hydrogen bonding, and sequence requirements of telomerase inhibition using the TRAP assay and a bioassay measuring cell viability following exposure to the compounds. In addition, we have also studied the role of the 3' end and secondary structure of telomere mimics on telomerase inhibition. Observations reveal that sensitivity to the S-ODNs may not require hybridization to an antisense target but required guanine nucleotides on the 3' end for cells in culture and telomerase inhibition in vitro. The importance of H bonding and the requirement for a free 3' end for the activity of these compounds has also been demonstrated. However, transient inhibition of telomerase is not cytotoxic to all immortal cells and is not sufficient to explain the mechanism of cytotoxicity of these short oligonucleotides.     

Nuclear organization of splicing small nuclear ribonucleoproteins in adenovirus-infected cells.

We have studied the effect of adenovirus infection on the nuclear organization of splicing small nuclear ribonucleoproteins (snRNPs) in HeLa cells. In uninfected HeLa cells, snRNPs are widespread throughout the nucleoplasm but also are concentrated in specific nuclear structures, including coiled bodies, interchromatin granules, and perichromatin fibrils. We have used immunofluorescence microscopy to study the localization of splicing snRNPs relative to centers of viral DNA synthesis and accumulation identified with antiserum against the viral 72,000-molecular-weight single-stranded DNA-binding protein (72K protein). Splicing snRNPs were independently detected with both monoclonal and polyclonal antibodies specific for common snRNP antigens, snRNP-specific proteins, and the snRNA-specific 2,2,7-trimethylguanosine 5' cap structure. We have examined infected cells 2 to 24 h after infection, and, in the majority of these cells, we observed no colocalization of the snRNP and 72K-protein staining patterns. In the late phase, snRNPs were found to markedly concentrate in discrete clusters that were distinct from the centers of viral DNA synthesis and accumulation identified with anti-72K protein. We have treated cells with hydroxyurea at various times after infection to inhibit aspects of the virus infectious program. We have found that the accumulation of snRNP clusters is correlated with late gene expression rather than with DNA synthesis or early gene expression. Finally, we show that the late-phase snRNP clusters colocalize with a monoclonal antibody that primarily stains interchromatin granules. These results suggest that the centers of snRNP concentration in late-phase infected cells are likely to correspond to interchromatin granule clusters.     

Advances in tracking small migratory birds: a technical review of light‐level geolocation

Light‐level geolocation data loggers, or geologgers, have recently been miniaturized to the extent that they can be deployed on small songbirds, allowing us to determine many previously unknown migration routes, breeding locations, and wintering sites. Use of geologgers on small birds has great potential to help address major research and conservation questions, but the method is not without its shortcomings. Among these shortcomings are the need to recapture birds after they have carried a device throughout a migration cycle and the potential for the devices to affect survival and behavior. We examined return rates of birds with geologgers in published and unpublished studies and found no evidence of a general negative effect of geologgers on survival, although there were a few individual studies where such an effect was evident. From these same studies, we found that most currently used harness materials are equivalent in terms of failure rates, and the most reliable geologgers are those made by the British Antarctic Survey (although these were also the largest geologgers used in the studies we examined). With regard to analysis methods, we believe there is much room for improvement. Use of online archiving of both data and analysis parameters would greatly improve the repeatability and transparency of geologger research.     

NAD(+) and axon degeneration revisited: Nmnat1 cannot substitute for Wld(S) to delay Wallerian degeneration

The slow Wallerian degeneration protein (Wld(S)), a fusion protein incorporating full-length nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1), delays axon degeneration caused by injury, toxins and genetic mutation. Nmnat1 overexpression is reported to protect axons in vitro, but its effect in vivo and its potency remain unclear. We generated Nmnat1-overexpressing transgenic mice whose Nmnat activities closely match that of Wld(S) mice. Nmnat1 overexpression in five lines of transgenic mice failed to delay Wallerian degeneration in transected sciatic nerves in contrast to Wld(S) mice where nearly all axons were protected. Transected neurites in Nmnat1 transgenic dorsal root ganglion explant cultures also degenerated rapidly. The delay in vincristine-induced neurite degeneration following lentiviral overexpression of Nmnat1 was significantly less potent than for Wld(S), and lentiviral overexpressed enzyme-dead Wld(S) still displayed residual neurite protection. Thus, Nmnat1 is significantly weaker than Wld(S) at protecting axons against traumatic or toxic injury in vitro, and has no detectable effect in vivo. The full protective effect of Wld(S) requires more N-terminal sequences of the protein.     

Taxonomic affinities of dark septate root endophytes of Colobanthus quitensis and Deschampsia antarctica,the two native Antarctic vascular plant species

Although dark septate fungal endophytes (DSE) occur widely in association with plant roots in cold-stressed habitats, little is known of the taxonomic status of DSE in Antarctica. Here we investigate the phylogenetic affinities of DSE colonising the roots of Colobanthus quitensis and Deschampsia antarctica, the two maritime Antarctic vascular plant species. Two hundred and forty-three DSE were isolated from roots collected from 17 sites across a 1 470 km transect through maritime and sub-Antarctica. The ITS1-5.8S-ITS2 nuclear ribosomal gene cluster of representative isolates was sequenced, and the sequences were recovered in 10 sequence groups and sub-groups. Nine of the sequence groupings could be placed in the Helotiales and the remaining one showed high homology to a large number of currently unassigned anamorphic ascomycete sequences. Of the Helotiales, Leptodontidium orchidicola, Rhizoscyphus ericae and species of Tapesia and Mollisia could be confidently identified. This study demonstrates that members of the Helotiales, including several widely-recognised DSE genera, commonly colonise the roots of C. quitensis and D. antarctica in the Antarctic.     

Sodium-Calcium Exchange Is Essential for Effective Triggering of Calcium Release in Mouse Heart

In cardiac myocytes, excitation-contraction coupling depends upon sarcoplasmic reticular Ca²⁺ release triggered by Ca²⁺ influx through L-type Ca²⁺ channels. Although Na⁺-Ca²⁺ exchange (NCX) is essential for Ca²⁺ extrusion, its participation in the trigger process of excitation-contraction coupling is controversial. To investigate the role of NCX in triggering, we examined Ca²⁺ sparks in ventricular cardiomyocytes isolated from wild-type (WT) and cardiac-specific NCX knockout (KO) mice. Myocytes from young NCX KO mice are known to exhibit normal resting cytosolic Ca²⁺ and normal Ca²⁺ transients despite reduced L-type Ca²⁺ current. We loaded myocytes with fluo-3 to image Ca²⁺ sparks using confocal microscopy in line-scan mode. The frequency of spontaneous Ca²⁺ sparks was reduced in KO myocytes compared with WT. However, spark amplitude and width were increased in KO mice. Permeabilizing the myocytes with saponin eliminated differences between spontaneous sparks in WT and KO mice. These results suggest that sarcolemmal processes are responsible for the reduced spark frequency and increased spark width and amplitude in KO mice. When myocytes were loaded with 1 mM fluo-3 and 3 mM EGTA via the patch pipette to buffer diadic cleft Ca²⁺, the number of sparks triggered by action potentials was reduced by 60% in KO cells compared to WT cells, despite similar SR Ca²⁺ content in both cell types. When EGTA was omitted from the pipette solution, the number of sparks triggered in KO and WT myocytes was similar. Although the number of sparks was restored in KO cells, Ca²⁺ release was asynchronous. These results suggest that high subsarcolemmal Ca²⁺ is required to ensure synchronous triggering with short spark latency in the absence of NCX. In WT mice, high subsarcolemmal Ca²⁺ is not required for synchronous triggering, because NCX is capable of priming the diadic cleft with sufficient Ca²⁺ for normal triggering, even when subsarcolemmal Ca²⁺ is lowered by EGTA. Thus, reducing subsarcolemmal Ca²⁺ with EGTA in NCX KO mice reveals the dependence of Ca²⁺ release on NCX.     

Cryptic plasticity underlies a major evolutionary transition

The origin of eusociality is often regarded as a change of macroevolutionary proportions [1, 2]. Its hallmark is a reproductive division of labor between the members of a society: some individuals ("helpers" or "workers") forfeit their own reproduction to rear offspring of others ("queens"). In the Hymenoptera (ants, bees, wasps), there have been many transitions in both directions between solitary nesting and sociality [2-5]. How have such transitions occurred? One possibility is that multiple transitions represent repeated evolutionary gains and losses of the traits underpinning sociality. A second possibility, however, is that once sociality has evolved, subsequent transitions represent selection at just one or a small number of loci controlling developmental switches between preexisting alternative phenotypes [2, 6]. We might then expect transitional populations that can express either sociality or solitary nesting, depending on environmental conditions. Here, we use field transplants to directly induce transitions in British and Irish populations of the sweat bee Halictus rubicundus. Individual variation in social phenotype was linked to time available for offspring production, and to the genetic benefits of sociality, suggesting that helping was not simply misplaced parental care [7]. We thereby demonstrate that sociality itself can be truly plastic in a hymenopteran.     

Ontogenetic variations in the venom proteome of the Amazonian snake Bothrops atrox

Background  

Bothrops atrox is responsible for the majority of snakebite accidents in the Brazilian Amazon region. Previous studies have demonstrated that the biological and pharmacological activities of B. atrox venom alter with the age of the animal. Here, we present a comparative proteome analysis of B. atrox venom collected from specimens of three different stages of maturation: juveniles, sub-adults and adults.