A genomic map enriched for markers linked to Avr1 in Cronartium quercuum f.sp. fusiforme

A novel approach is presented to map avirulence gene Avr1 in the basidiomycete Cronartium quercuum f.sp. fusiforme, the causal agent of fusiform rust disease in pines. DNA markers tightly linked to resistance gene Fr1 in loblolly pine tree 10-5 were used to classify 10-5 seedling progeny as either resistant or susceptible. A single dikaryotic isolate (P2) heterozygous at the corresponding Avr1 gene was developed by crossing Fr1 avirulent isolate SC20-21 with Fr1 virulent isolate NC2-40. Bulk basidiospore inoculum derived from isolate P2 was used to challenge the pine progeny. The ability to unambiguously marker classify 10-5 progeny as resistant (selecting for virulence) or susceptible (non-selecting) permitted the genetic mapping of the corresponding Avr1 gene by bulked segregant analysis. Using this approach, 14 genetic markers significantly linked to Avr1 were identified and placed within the context of a genome-wide linkage map produced for isolate P2 using samples from susceptible seedlings.     

mTOR drives its own activation via SCF(βTrCP)-dependent degradation of the mTOR inhibitor DEPTOR

The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCF(βTrCP). DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway might contribute to aberrant activation of mTOR in disease.     

Evidence for the presence of a critical disulfide bond in the mouse EP3γ receptor

To determine the contribution of cysteines to the function of the mouse E-prostanoid subtype 3 gamma (mEP3γ), we tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from HEK293 cells transfected with mEP3γ C107A or C184A had no detectable radioligand binding. Other mutant mEP3γ receptors had radioligand affinities and receptor densities similar to wild-type. Cell-surface ELISA against the N-terminal HA-tag of C107A and C184A demonstrated 40% and 47% reductions respectively in receptor protein expression at the cell surface, and no radioligand binding was detected as assessed by intact cell radioligand binding experiments. These data suggest a key role for C107 and C184 in both receptor structure/stability and function and is consistent with the presence of a conserved disulfide bond between C107 and C184 in mouse EP3 that is required for normal receptor expression and function. Our results also indicate that if a second disulfide bond is present in the native receptor it is non-essential for receptor assembly or function.     

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit. To more fully characterize this family of enzymes, we have investigated the substrate requirements of ArmA and to a lesser extent its ortholog RmtA. We determined the Mg+2 dependence of ArmA activity toward the 30S ribosomal subunits and found that the enzyme recognizes both low Mg+2 (translationally inactive) and high Mg+2 (translationally active) forms of this substrate. We tested the effects of LiCl pretreatment of the 30S subunits, initiation factor 3 (IF3), and gentamicin/kasugamycin resistance methyltransferase (KsgA) on ArmA activity and determined whether in vivo derived pre-30S ribosomal subunits are ArmA methylation substrates. ArmA failed to methylate the 30S subunits generated from LiCl washes above 0.75 M, despite the apparent retention of ribosomal proteins and a fully mature 16S rRNA. From our experiments, we conclude that ArmA is most active toward the 30S ribosomal subunits that are at or very near full maturity, but that it can also recognize more than one form of the 30S subunit.     

Aging and factors related to running economy

The purpose of this study was to investigate the relationship that age has on factors affecting running economy (RE) in competitive distance runners. Fifty-one male and female subelite distance runners (Young [Y]: 18-39 years [n = 18]; Master [M]: 40-59 years [n = 22]; and Older [O]: 60-older [n = 11]) were measured for RE, step rate, lactate threshold (LT), VO2max, muscle strength and endurance, flexibility, power, and body composition. An RE test was conducted at 4 different velocities (161, 188, 215, and 241 m·min(-1)), with subjects running for 5 minutes at each velocity. The steady-state VO2max during the last minute of each stage was recorded and plotted vs. speed, and a regression equation was formulated. A 1 × 3 analysis of variance revealed no differences in the slopes of the RE regression lines among age groups (y = 0.1827x - 0.2974; R2 = 0.9511 [Y]; y = 0.1988x - 1.0416; R2 = 0.9697 [M]; y = 0.1727x + 3.0252; R2 = 0.9618 [O]). The VO2max was significantly lower in the O group compared to in the Y and M groups (Y = 64.1 ± 3.2; M = 56.8 ± 2.7; O = 44.4 ± 1.7 mlO2·kg(-1)·min(-1)). The maximal heart rate and velocity @ LT were significantly different among all age groups (Y = 197 ± 4; M = 183 ± 2; O = 170 ± 6 b·min(-1) and Y = 289.7 ± 27.0; M = 251.5 ± 32.9; O = 212.3 ± 24.6 m·min(-1), respectively). The VO2max @ LT was significantly lower in the O group compared to in the Y and M groups (Y = 50.3 ± 2.0; M = 48.8 ± 2.9; O = 34.9 ± 3.2 mlO2·kg(-1)·min(-1)). The O group was significantly lower than in the Y and M groups in flexibility, power, and upper body strength. Multiple regression analyses showed that strength and power were significantly related to running velocity. The results from this cross-sectional analysis suggest that age-related declines in running performance are associated with declines in maximal and submaximal cardiorespiratory variables and declines in strength and power, not because of declines in running economy.     

Upper-body anthropometric and strength measures and their relationship to start time in elite luge athletes

Start time has been shown to be a significant predictor of overall performance in the sport of luge. The starting motion in luge has been described as an explosive upper-body movement that requires significant technique and skill to perfect. This study aims to investigate upper-body factors that may relate to start time in luge. Twenty-two subjects participated in the study as part of their normal off-season training. Each subject had a minimum of 3 years' experience in the sport of luge, and at the time was a member of a U.S. Luge National Team. Subjects completed a 1 repetition maximum (1RM) in the bench press (BP), prone row (PR), and weighted pull-up (WP). Anthropometric distances were taken measuring finger-tip span (FS), biacromial breadth (BB), acromio-radial length, acromio-olecranon length (AO), hand length, and sitting cervical height. Subjects were divided into 2 groups based on which U.S. Luge National team they were currently a member of, Senior National (SN, n = 13) and Junior National (JN, n = 9). A Pearson product-moment correlation coefficient showed several significant (p ≤ 0.05) relationships between upper-body variables and start time among the groups. The BP and PR 1RM were shown to have a significant relationship in both groups. Among the anthropometric variables, AO was also significant in both groups. The WP, FS, BB, and height were all shown to have a significant relationship with start time in the SN group, but not in the JN group. These results suggest that as luge athletes become more efficient in the starting technique, outside factors such as upper-body strength and anthropometric measures play a larger role in performance.     

The effects of a constant sprint-to-rest ratio and recovery mode on repeated sprint performance

It is unclear if a constant sprint-to-rest ratio allows full performance recovery between repeated sprints over different distances. This is important for the development of sprint-training programs. Additionally, there is conflicting evidence on whether active recovery enhances sprint performance. Three repeated sprint protocols were used (22 × 15, 13 × 30, and 8 × 50 m), with each having an active and passive recovery. Each trial was conducted with an initial sprint-to-rest ratio of 1:10. Repeated sprints were analyzed by comparing the first sprint to the last sprint. For the 15-m trials, there were no significant main effects for recovery or time and no significant interaction. For the 30-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 15.995, p = 0.003; mean difference = 0.20 seconds, 95% confidence interval [CI] = 0.09-0.31 seconds, d = 1.4 [large effect]). There was no interaction of recovery and time in the 30-m trials. For the 50-m trials, there was no main effect for recovery, but a main effect for time (F[1,10] = 34.225, p = 0.0002; mean difference = 0.39 seconds, 95% CI = 0.24-0.55 seconds, d = 1.3 [large effect]). There was no interaction of recovery and time in the 50-m trials. The results demonstrate that a 1:10 sprint-to-rest ratio allows full performance recovery between 15-m sprints, but not between sprints of 30 or 50 m, and that recovery mode did not influence repeated sprint performance.     

Spatial patterns of variation in color and spine shape in the sea urchin Heliocidaris erythrogramma

Abstract. Here we report on the first quantitative survey of morphological variation in the sea urchin Heliocidaris erythrogramma within Western Australia and distinguish between two subspecies found to co‐occur in this region. We surveyed urchins at multiple spatial scales along the Western Australian coastline to assess variation in dermis and spine color and, using landmark‐based geometric morphometrics, spine morphology. Both color and morphology proved to be useful for separating subspecies within Western Australia. There were four major color morphs: red dermis/violet spines (56%), red/violet‐green (23%), red/green (7%), and white/green (10%). Members of the first two color morphs had bulbous spines with wide, flattened tips, a morphology that is unique to Western Australia and characteristic of H. e. armigera, and members of the latter two consistently exhibited the narrow, pointed spines typical of specimens of H. e. erythrogramma, which has a broader distribution. In Western Australia, H. e. armigera was relatively abundant both within and among sites, but H. e. erythrogramma was found only in a few localized patches. Shifts in the relative abundance of these two subspecies occurred at fine spatial scales (<5 km), although environmental correlates of these transitions were unclear. Contrary to expectations, neither dermis color nor spine morphology varied with relative wave exposure: individuals with a red dermis or thickened spine morphology occurred at most sites regardless of exposure, and while white dermis and thinner spines only occurred at high‐exposure sites, these features were not common across the majority of exposed sites. Both color morph frequencies and spine morphology remained stable within sites over the 3‐year duration of this study. While the ecological significance of this morphological variation remains unclear, the consistency of the association between color and spine morphology, occurring across fine spatial scales, suggests that strong environmental or genetic factors are involved in maintaining morphological differentiation between these two subspecies.     

The structure of a COPII tubule

Nearly a third of all eukaryotic proteins are transported from the ER to the Golgi apparatus through the secretory pathway using COPII coated vesicles. Evidence suggests that this transport occurs via 500–900 Å vesicles that bud from the ER membrane. It has been shown that procollagen molecules utilize the COPII proteins for transport, but it is unclear how the COPII coat can accommodate these ～3000 Å long molecules. We now present a cryogenic electron tomographic reconstruction of a Sec13/31 tubule that is approximately 3300 Å long containing a hollow cylindrical interior that is 300 Å in diameter, dimensions that are consistent with those that are required to encapsulate a procollagen molecule wrapped in a membrane and accessory COPII components. This structure suggests a novel mechanism that the COPII coat may employ to transport elongated cargo.     

The ribonucleotide reductase R1 subunits of herpes simplex virus types 1 and 2 protect cells against TNFα- and FasL-induced apoptosis by interacting with caspase-8

We previously reported that HSV-2 R1, the R1 subunit (ICP10; UL39) of herpes simplex virus type-2 ribonucleotide reductase, protects cells against apoptosis induced by the death receptor (DR) ligands tumor necrosis factor-alpha- (TNFα) and Fas ligand (FasL) by interrupting DR-mediated signaling at, or upstream of, caspase-8 activation. Further investigation of the molecular mechanism underlying HSV-2 R1 protection showed that extracellular-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3-K)/Akt, NF-κB and JNK survival pathways do not play a major role in this antiapoptotic function. Interaction studies revealed that HSV-2 R1 interacted constitutively with caspase-8. The HSV-2 R1 deletion mutant R1(1-834)-GFP and Epstein–Barr virus (EBV) R1, which did not protect against apoptosis induced by DR ligands, did not interact with caspase-8, indicating that interaction is required for protection. HSV-2 R1 impaired caspase-8 activation induced by caspase-8 over-expression, suggesting that interaction between the two proteins prevents caspase-8 dimerization/activation. HSV-2 R1 bound to caspase-8 directly through its prodomain but did not interact with either its caspase domain or Fas-associated death domain protein (FADD). Interaction between HSV-2 R1 and caspase-8 disrupted FADD-caspase-8 binding. We further demonstrated that individually expressed HSV-1 R1 (ICP6) shares, with HSV-2 R1, the ability to bind caspase-8 and to protect cells against DR-induced apoptosis. Finally, as the long-lived Fas protein remained stable during the early period of infection, experiments with the HSV-1 UL39 deletion mutant ICP6∆ showed that HSV-1 R1 could be essential for the protection of HSV-1-infected cells against FasL.