297 research outputs found

    Tti2 in PIKK Biosynthesis and Its Use in Identifying Missense Suppressor tRNAs

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    Protein biosynthesis is an essential process for all cells. It involves the translation of genetically encoded information into peptides, folding and assembly of peptides into three-dimensional molecules and complexes, and post-translational modification. Molecular chaperones facilitate protein folding so that a native state is achieved. Misfolded proteins and aggregates are toxic within the cell and accumulate due to stress conditions, mutations, and cell aging. Moreover, essential proteins rely on chaperones and co-chaperones for their regulation and activity. The TTT complex, consisting of Tel2, Tti1, and Tti2, is considered an Hsp90 co-chaperone with specificity for phosphatidylinositol 3-kinase-related kinases (PIKKs). I show that yeast expressing low levels of Tti2 are viable under optimal growth conditions, but are sensitive to stress conditions that involve PIKK pathways. In agreement with this, depleting Tti2 levels decreased expression of the PIKK proteins Tra1, Mec1, and Tor1, and affected their localization. I also find that overexpressing Hsp90 or its co-chaperones is synthetic lethal when Tti2 is depleted, an effect possibly due to imbalanced stoichiometry of a complex required for PIKK assembly. These results indicate that Tti2 does not act as a general chaperone, but may have a specialized function in PIKK folding and/or complex assembly. Using a stress sensitive tti2 allele with a Leu to Pro mutation at residue 187, I performed a genetic selection of spontaneous second site mutations. The selection identified a single nucleotide mutation at the same position (C70T) in four tRNAProUGG genes. Since the mutation incorporates the identity element (a G3:U70 base pair) for alanyl-tRNA synthetase into the tRNAPro acceptor stem, I hypothesized that suppression results from mistranslation of Pro187 in Tti2L187P as Ala. In vitro, tRNAProUGG (C70U) was mis-aminoacylated with alanine by alanyl-tRNA synthetase. Mass spectrometry from protein expressed in vivo and a novel GFP reporter for mistranslation confirmed substitution of alanine for proline at a rate of ~6%. Mistranslating cells expressing tRNAProUGG (C70U) induce a partial heat shock response but grow nearly identically to wild-type

    Phenotypic Analysis of Schizosaccharomyces Pombe Strains bearing Site-Directed Mutations in the Carboxy Terminal Domain of the Largest Subunit of RNA Polymerase II

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    The phosphorylation status of the largest sub-unit of RNA polymerase II (Rpb1p) is crucial to the control of transcription in eukaryotes. The domain subject to this phosphorylation is known as the carboxyl terminal domain (CTD) and consists of multiple repeats (from 20 to 52 copies depending on the species in question) of the heptad sequence Y1S2P3T4S5P6S7. Interestingly, differential phosphorylation of S2, S5, and S7 residues is known to play an important role in the control of pre-mRNA processing. To determine the number of CTD repeats required for viability in Schizosaccharomyces pombe, truncated CTD constructs were integrated into the S. pombe genome through homologous recombination. I have observed that a minimum of eight heptad repeats is required for normal growth. While five heptad repeats is sufficient for viability, strains bearing these constructs exhibit impaired growth as well as morphological abnormalities. Next, in order to study the phenotypic effects of altered phosphorylation patterns, site-directed mutants were created in which alanine residues replaced serine (mimicking the non-phosphorylated state) or in which glutamate residues replaced serine (mimicking the phosphorylated state). Interestingly, alteration of the phosphorylation status of both S5 and S7 residues resulted in a variety of pleiotropic defects related to both cytokinesis and morphogenesis. Such a role for S5 and S7 phosphorylation is further supported by genetic analysis demonstrating synthetic genetic interactions between CTD site mutants and mutants affecting the function of the cell division machinery

    A Deployment of Spreadsheets

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    Many steganographers would agree that, had it not been for the appropriate unification of the partition table and cache coherence, the unproven unification of IPv7 and architecture might never have occurred. In fact, few researchers would disagree with the evaluation of evolutionary programming. Our focus here is not on whether the much-touted replicated algorithm for the deployment of forward-error correction by Gupta and White runs in Ω(n) time, but rather on presenting a semantic tool for deploying operating systems (Taw)

    Spatial Awareness is Related to Moderate Intensity Running during a Collegiate Rugby Match

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    International Journal of Exercise Science 9(5): 599-606, 2016. The purpose of the present study was to evaluate the relationship between spatial awareness, agility, and distance covered in global positioning system (GPS) derived velocity zone classifications during a collegiate rugby match. Twelve American collegiate rugby union players (mean±SD; age: 21.2±1.4 y; weight: 85.0±16.0 kg; 7 forwards & 5 backs) on a single team volunteered to participate in this investigation. The distances travelled at low (walking/jogging; \u3c2.7m/s), moderate (cruising/striding; 2.7-5.0 m/s), and high intensities (running/sprinting; \u3e5.0 m/s) were measured for each player using GPS sensors and normalized according to playing time during an official USA Rugby match. Spatial awareness was measured as visual tracking speed from one core session of a 3-dimensional multiple-object-tracking speed (3DMOTS) test (1.35±0.59 cm·sec-1). Agility was assessed utilizing the pro agility (5.05±0.28 sec) and t drill (10.62±0.39 sec). Analysis of variance revealed that athletes travelled the greatest distance during walking/jogging (39.5±4.5 m·min-1) and least distance during running/sprinting (4.9±3.5 m·min-1). Pearson product moment correlations revealed that only distance covered while cruising/striding (20.9±6.5 m·min-1) was correlated to spatial awareness (r=0.798, p=0.002). Agility did not correlate to distance covered at any velocity zone or spatial awareness. Spatial awareness, as determined by 3DMOTS, appears to be related to the moderate intensity movement patterns of rugby union athletes

    Biomarkers of muscle quality: N-terminal propeptide of type III procollagen and C-terminal agrin fragment responses to resistance exercise training in older adults

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    Background N-terminal peptide of procollagen type III (P3NP) and C-terminal agrin fragment (CAF) are circulating biomarkers that are related to lean body mass in older adults. P3NP is a circulating marker reflective of muscular structural remodeling while CAF is a circulating marker of neuromuscular remodeling. As resistance exercise is an established intervention that can effectively improve muscle quality, we sought to evaluate circulating biomarker changes corresponding to a resistance exercise intervention in older adults. Methods Twenty-three older adults (aged 61 to 85 years) were randomized into an intervention (6-week resistance training) or control group. Resting circulating P3NP, CAF, lean body mass (LBM), muscle cross-sectional area (CSA), muscle strength, and muscle quality were determined at baseline and after the intervention or control period by enzyme-linked immunosorbent assay, dual-energy X-ray absorptiometry, ultrasound, leg extension, and relative strength, respectively. Changes in circulating biomarkers and measures of muscle mass and quality were evaluated with repeated-measures analysis of variance; clinical interpretations were made with magnitude-based inferences, and relationships between variables were evaluated with bivariate correlations. Results The short-term resistance exercise intervention was effective at improving muscle quality by 28 % (p \u3c 0.001) despite no significant changes in lean body mass. Baseline circulating P3NP was somewhat lower in older women (4.15 +/- 1.9 ng/mL) compared with older men (4.81 +/- 2.1 ng/mL). The exercise intervention tended to increase circulating P3NP (baseline=4.53 + 1.80 to post=4.88 + 1.86) and was significantly correlated with changes in LBM (r=0.422, p=0.045). At baseline, women (3.91 + 1.12 pg/mL) had somewhat higher circulating CAF than men (3.47 +/- 1.37 pg/mL). Circulating CAF increased by 10.4 % (3.59 to 4.00 pg/ml) in older adults following 6 weeks of resistance exercise training. Changes in circulating CAF were significantly related to changes in CSA of the vastus lateralis (r=0.542, p=0.008). Conclusions Assessment of P3NP and CAF from blood samples may provide minimally invasive and clinically informative measures of skeletal muscle status in older adults. Circulating CAF appears to increase in response to short-term resistance exercise training in older adults to a clinically meaningful magnitude. Changes in circulating P3NP in response to the intervention were less clear but appear to reflect muscle hypertrophy. Further research is needed to elucidate whether P3NP, CAF, or other biomarkers can reflect muscle qualitative adaptations with larger and longer studies

    Reliability of the Dynavision (TM) D2 for Assessing Reaction Time Performance

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    Recently, the Dynavision (TM) D2 Visuomotor Training Device (D2) has emerged as a tool in the assessment of reaction time (RT); however, information regarding the reliability of the D2 have been limited, and to date, reliability data have been limited to non-generalizable samples. Therefore, the purpose of this study was to establish intraclass correlation coefficients (ICC2,1) for the D2 that are generalizable across a population of recreationally active young adults. Forty-two recreationally active men and women (age: 23.41 +/- 4.84 years; height: 1.72 +/- 0.11 m; mass: 76.62 +/- 18.26 Kg) completed 6 trials for three RT tasks of increasing complexity. Each trial was separated by at least 48-hours. A repeated measures ANOVA was used to detect differences in performance across the six trials. Intraclass correlation coefficients (ICC2,1) standard error of measurement (SEM), and minimal differences (MD) were used to determine the reliability of the D2 from the two sessions with the least significant difference score. Moderate to strong reliability was demonstrated for visual RT (ICC2,1: 0.84, SEM: 0.033), and reactive ability in both Mode A and Mode B tasks (Mode A hits: ICC2,1: 0.75, SEM: 5.44; Mode B hits: ICC2,1: 0.73, SEM: 8.57). Motor RT (ICC2,1: 0.63, SEM: 0.035s) showed fair reliability, while average RT per hit for Modes A and B showed moderate reliability (ICC2,1: 0.68, SEM: 0.43 s and ICC2,1: 0.72, SEM: 0.03 s respectively). It appears that one familiarization trial is necessary for the choice reaction time (CRT) task while three familiarization trials are necessary for reactive RT tasks. In conclusion, results indicate that the Dynavision (TM) D2 is a reliable device to assess neuromuscular reactivity given that an adequate practice is provided. The data presented are generalizable to a population of recreationally active young adults

    Effects of b-hydroxy-bmethylbutyrate free acid and cold water immersion on postexercise markers of muscle damage.

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    Abstract The aim of the current study was to examine the effects of cold water immersion (CWI) with and without the free acid form of b-hydroxy-b-methylbutyrate (HMB-FA) on markers of muscle damage following acute lower body resistance exercise. Forty recreationally resistance-trained men (22.3 ± 2.4 years) were randomly divided into one of the four groups: (1) Placebo (PL); (2) HMB-FA; (3) HMB-FA-CWI; (4) PL-CWI. HMB-FA groups ingested 3 g day -1 and CWI groups submersed their lower body into 10-12°C water for 10-min postexercise. No differences between groups were observed for CK; however, PL-CWI had significantly greater elevations in myoglobin 30-min post-exercise compared to HMB-FA (p = 0.009) and PL (p = 0.005), and HMB-FA-CWI was significantly greater than HMB-FA (p = 0.046) and PL (p = 0.028). No differences between groups were observed for IL-6 and IL-10, although CRP was significantly greater 24-h post-exercise for PL-CWI compared to HMB-FA-CWI (p = 0.02) and HMB-FA (p = 0.046). Only HMB-FA-CWI showed significantly (p = 0.02) greater improvements in average power per repetition. CWI appeared to elevate myoglobin compared to other groups, while HMB-FA may have attenuated the increase in CRP when combined with CWI. Nevertheless, HMB-FA or CWI treatments did not appear to provide benefit over PL for recovery. Instead, the combination of CWI and HMB-FA improved performance recovery compared to other groups
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