The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction

Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p. Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length. To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths. While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences. We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites. As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement. The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation. These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.     

Deformable dielectrics – optimization of heterogeneities

The modeling and the numerical simulation of deformable dielectrics are discussed in a coupled electro-mechanical framework. It is demonstrated how the Maxwell stress contributions can be implemented efficiently into a finite element method. By using numerical simulation, the electro-static contraction of a soft actuator is calculated and examined. The operational curve, i.e. the variation of the compression versus the applied electric field, is obtained for a capacitor-like setup using the neo-Hooke material model. Furthermore, the idea of introducing ceramic inclusions in the soft elastomer is discussed, which is shown to be capable of improving the efficiency of the actuator. By representative examples, the geometry optimization of the inclusion is considered.     

Photophobia and phonophobia in migraineurs between attacks

This study investigated whether migraineurs are more sensitive to light and sound while headache-free than are healthy people. Fifty-two migraineurs (mean age 39 years) were selected using the International Headache Society diagnostic criteria for migraine. Forty-eight healthy controls were matched for age, sex, and race (mean age 36 years). Visual and auditory discomfort thresholds were measured by exposing subjects to increasing light and sound until they complained of discomfort. There were significant differences between groups in both the light discomfort threshold (P < 0.00005) and the hearing discomfort threshold (P < 0.0005). The thresholds for both were lower in the migraineurs. Overall, for both groups together, there was a significant negative correlation between light discomfort threshold and age (correlation coefficient -0.2276, P = 0.011), but not for the hearing discomfort threshold and age (P = 0.275). The results show that the migraineurs were significantly more sensitive to light and sound when headache-free than were healthy controls. The apparent increased intolerance to light in both groups together noted with increased age, did not apply to the migraine group.     

trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP

Simian virus 40 (SV40) DNA replication requires the coordinated action of multiple biochemical activities intrinsic to the virus-encoded large tumor antigen (T antigen). We report the preliminary biochemical characterization of the T antigens encoded by three SV40 mutants, 5030, 5031, and 5061, each of which have altered residues within or near the ATP binding pocket. All three mutants are defective for viral DNA replication in cultured cell lines. However, while 5030 and 5031 can be complemented in vivo by providing a wild-type T antigen in trans, 5061 exhibits a strong trans-dominant-negative phenotype. In order to determine the basis for their replication defects and to explore the mechanisms of trans dominance, we purified the T antigens encoded by each of these mutants and examined their activities in vitro. The 5061 T antigen had no measurable ATPase activity and failed to hexamerize in response to ATP, and its affinity for the SV40 origin of DNA replication (ori) DNA was not increased by ATP. In contrast, the 5030 and 5031 T antigens exhibited at least some ATPase activity and both readily formed hexamers in the presence of ATP. These mutants differed in that 5030 was very defective in an ori-dependent unwinding assay while 5031 retained significant activity. Both the 5030 and 5031 T antigens bound to ori-containing DNA, but the binding was less efficient than that of wild-type T antigen and was not affected by the presence of ATP. These results suggest that 5030 and 5031 are defective in some aspect of communication between the ATP binding and DNA binding domains and that the ability of ATP to induce T-antigen hexamerization is distinct from its action to increase the affinity for ori. Finally, all three mutants were defective for the ability to support SV40 DNA replication in vitro. Both the 5031 and 5061 T antigens inhibited wild-type-T-antigen-stimulated replication in vitro, while the 5030 T antigen did not. The fact that the 5031 T antigen was trans dominant in the in vitro assays but not in vivo indicates that the in vitro system does not accurately reflect events occurring in vivo.     

Numerical Simulation of a Mixed-mode Dynamic Fracture Experiment

A time-domain boundary element method is applied to simulate mixed-mode dynamic fracture experiments performed on compact compression specimens (CCS) in a split Hopkinson bar. This numerical investigation addresses the time histories of the mode I and II stress intensity factors for the precracked CCS, the instant and angle of crack initiation as well as the resulting crack paths in dependence upon the external load. The aim of the study is two-fold: to gain deeper insight into the mixed-mode dynamic fracture process under investigation and to test the applicability of our recently developed numerical method. Numerical results are compared to experimental data reported in the literature. Furthermore, aspects of modelling the experimental set-up in the framework of the boundary element method are discussed, and advantages and shortcomings are analyzed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).     

Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe

Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1. We first constructed S. pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine. This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent. These mutants, however, grow normally when Ras1 is expressed (ras1+). These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S. pombe.     

Medium Scale Gravity Waves in the Thermosphere Observed by the AE-C Satellite

Atmospheric Explorer C (AE-C) satellite data were used to investigate the spectral characteristics of medium-scale wavelike structures (horizontal-scale size from tens to hundreds of kilometers) observed in the neutral and ionized components of the thermosphere at about 250-km altitude. Data for the neutral densities of oxygen, nitrogen, and helium from the Neutral Atmosphere Temperature Experiment, the electron density and temperature from the Cylindrical Electrostatic Probe were analyzed for a part of an orbit in the northern hemisphere between 54° and 10° geographic latitudes. Coherent wave systems are shown by employing direct comparison of the fluctuations of the parameters, comnputations of their cross-correlation functions and spectral analysis. Waves from many tens to hundreds of kilometers are found in the fluctuations of all parameters. A power law decrease of the spectrum is also found with different rates for the various densities and electron temperature, -3.2 for oxygen, -4.6 for nitrogen, 0.23 for helium, -1.96 for the electron density, and -1.66 for the electron temperature. These results complement similar studies of large-scale structures reported in another paper [8]. They are typical of those obtained for other parts of this orbit and for other orbits. Unlike largescale waves, medium-scale waves appear to attenuate after about 4-5 cycles, consistent with lower altitude sources and known loss mechanisms. An exception is shown with wave structure throughout the entire time record, however, implying a source in the F region.