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Crystal structure of the RUN domain of the RAP2-interacting protein x.

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 A of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel alpha-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.

Amino Acid Sequence↗

Statistical analysis of nucleotide runs in coding and noncoding DNA sequences.

A statistical analysis of the occurrence of particular nucleotide runs in DNA sequences of different species has been carried out. There are considerable differences of run distributions in DNA sequences of procaryotes, invertebrates and vertebrates. There is an abundance of short runs (1-2 nucleotides long) in the coding sequences and there is a deficiency of such runs in the noncoding regions. However, some interesting exceptions from this rule exist for the run distribution of adenine in procaryotes and for the arrangement of purine-pyrimidine runs in eucaryotes. The similarity in the distributions of such runs in the coding and noncoding regions may be due to some structural features of the DNA molecule as a whole. Runs of guanine (or cytosine) of three to six nucleotides occur predominantly in noncoding DNA regions in eucaryotes, especially in vertebrates.

Animals↗

Sequence composition and context effects on the generation and repair of frameshift intermediates in mononucleotide runs in Saccharomyces cerevisiae.

DNA polymerase slippage occurs frequently in tracts of a tandemly repeated nucleotide, and such slippage events can be genetically detected as frameshift mutations. In long mononucleotide runs, most frameshift intermediates are repaired by the postreplicative mismatch repair (MMR) machinery, rather than by the exonucleolytic proofreading activity of DNA polymerase. Although mononucleotide runs are hotspots for polymerase slippage events, it is not known whether the composition of a run and the surrounding context affect the frequency of slippage or the efficiency of MMR. To address these issues, 10-nucleotide (10N) runs were inserted into the yeast LYS2 gene to create +1 frameshift alleles. Slippage events within these runs were detected as Lys(+) revertants. 10G or 10C runs were found to be more unstable than 10A or 10T runs, but neither the frequency of polymerase slippage nor the overall efficiency of MMR was greatly influenced by sequence context. Although complete elimination of MMR activity (msh2 mutants) affected all runs similarly, analyses of reversion rates in msh3 and msh6 mutants revealed distinct specificities of the yeast Msh2p-Msh3p and Msh2p-Msh6p mismatch binding complexes in the repair of frameshift intermediates in different sequence contexts.

Aldehyde Oxidoreductases↗

Acute effects of treadmill running on lipoprotein(a) levels in males and females.

This investigation examined the acute response of serum lipoprotein(a) (Lp(a)) concentration immediately after, and during several days following, level and downhill motorized treadmill running. Eight males ran for 1 h on a level motorized treadmill at an intensity producing 90% maximum heart rate (MHR). On a separate occasion, three males and three females performed downhill (negative 13.4% incline) treadmill running at an intensity producing 75-80% MHR. For both protocols, serial blood samples were taken pre- and post-exercise and at the same time of day 1, 3, 5, and 7 days following exercise. Levels of Lp(a), creatine kinase (CK), C-reactive protein (CRP), and ferritin were measured. Repeated measures statistical analysis (Friedman ANOVA) showed no significant change in the median level of Lp(a) (level run, 5.0 mg.dl-1; downhill run, 7.45 mg.dl-1) across time following either protocol. After level running, ferritin levels 5 and 7 d post-exercise were significantly (P < 0.05) lower compared with immediately and 1 d post-exercise measures (Friedman ANOVA). Following level running, the Wilcoxon signed rank test showed significant (P < 0.05) elevations in CK levels immediately, 1 and 5 d post-exercise compared with pre-exercise values. Following downhill running. CK level was significantly elevated up to 3 d post-exercise (Wilcoxon signed rank). Calculated plasma volume did not change significantly following either protocol. These data suggest that Lp(a) does not change acutely in response to level or downhill treadmill running up to 60 min duration.

Acute-Phase Proteins↗

Run-down of the GABAA response under experimental ischaemia in acutely dissociated CA1 pyramidal neurones of the rat.

1. The effect of experimental ischaemia on the response to gamma-aminobutyric acid (GABA) was assessed in acutely dissociated CA1 pyramidal neurones of rats, using the patch-clamp technique. 2. Rapid application of 3 x 10(-5) M GABA induced a bicuculline-sensitive inward Cl- current (IGABA) at a holding potential (Vh) of -44 mV. The peak amplitude of IGABA showed a time-dependent decrease (run-down) when it was recorded with the conventional whole-cell mode without internal ATP. The run-down was not observed when the intracellular ATP concentration ([ATP]i) was maintained by the nystatin-perforated recording with an intracellular Na+ concentration ([Na+]i) of 0 mM. 3. When [Na+]i was increased to more than 30 mM, the IGABA run-down was observed even with the nystatin-perforated recording. 4. The IGABA run-down observed at 60 mM [Na+]i with the nystatin method was further enhanced under experimental ischaemia without changes in the reversal potential of IGABA. The enhanced run-down was suppressed by application of the Na+,K(+)-ATPase inhibitors, ouabain and SPAI-1. 5. IGABA run-down during ischaemia was also accompanied by an outward holding current and a concomitant increase in intracellular free Ca2+ concentration ([Ca2+]i) in 48.5% of the neurones. The outward current was a Ca(2+)-activated K+ current, which was blocked by 3 x 10(-7) M charybdotoxin. 6. In the inside-out mode of the single-channel analysis, GABA activated three subconductance states with conductances of 33.4, 22.7 and 15.2 pS. Reduction of ATP concentration from 2 to 0 mM on the intracellular side suppressed the channel activities, while an increase in Ca2+ concentration from 0.7 x 10(-9) to 1.1 x 10(-6) M had no effect. 7. These results suggest that ischaemia induces the run-down of the postsynaptic GABA response at the GABAA receptor level, and that this run-down is triggered by a decrease in [ATP]i.

Adenosine Triphosphate↗

Expression pattern, regulation, and biological role of runt domain transcription factor, run, in Caenorhabditis elegans.

The Caenorhabditis elegans run gene encodes a Runt domain factor. Runx1, Runx2, and Runx3 are the three known mammalian homologs of run. Runx1, which plays an essential role in hematopoiesis, has been identified at the breakpoint of chromosome translocations that are responsible for human leukemia. Runx2 plays an essential role in osteogenesis, and inactivation of one allele of Runx2 is responsible for the human disease cleidocranial dysplasia. To understand the role of run in C. elegans, we used transgenic run::GFP reporter constructs and a double-stranded RNA-mediated interference method. The expression of run was detected as early as the bean stage exclusively in the nuclei of seam hypodermal cells and lasted until the L3 stage. At the larval stage, expression of run was additionally detected in intestinal cells. The regulatory elements responsible for the postembryonic hypodermal seam cells and intestinal cells were separately located within a 7.2-kb-long intron region. This is the first report demonstrating that an intron region is essential for stage-specific and cell type-specific expression of a C. elegans gene. RNA interference analysis targeting the run gene resulted in an early larva-lethal phenotype, with apparent malformation of the hypodermis and intestine. These results suggest that run is involved in the development of a functional hypodermis and gut in C. elegans. The highly conserved role of the Runt domain transcription factor in gut development during evolution from nematodes to mammals is discussed.

Animals↗

Skeletal muscle adaptations in response to voluntary wheel running in myosin heavy chain null mice.

10.1152/ japplphysiol.00832.2001.-To examine the effects of gene inactivation on the plasticity of skeletal muscle, mice null for a specific myosin heavy chain (MHC) isoform were subjected to a voluntary wheel-running paradigm. Despite reduced running performance compared with nontransgenic C57BL/6 mice (NTG), both MHC IIb and MHC IId/x null animals exhibited increased muscle fiber size and muscle oxidative capacity with wheel running. In the MHC IIb null animals, there was no significant change in the percentage of muscle fibers expressing a particular MHC isoform with voluntary wheel running at any time point. In MHC IId/x null mice, wheel running produced a significant increase in the percentage of fibers expressing MHC IIa and MHC I and a significant decrease in the percentage of fibers expressing MHC IIb. Muscle pathology was not affected by wheel running for either MHC null strain. In summary, despite their phenotypes, MHC null mice do engage in voluntary wheel running. Although this wheel-running activity is lessened compared with NTG, there is evidence of distinct patterns of muscle adaptation in both null strains.

Adaptation, Physiological↗

Spike-wave complexes and fast components of cortically generated seizures. IV. Paroxysmal fast runs in cortical and thalamic neurons.

In the preceding papers of this series, we have analyzed the cellular patterns and synchronization of neocortical seizures occurring spontaneously or induced by electrical stimulation or cortical infusion of bicuculline under a variety of experimental conditions, including natural states of vigilance in behaving animals and acute preparations under different anesthetics. The seizures consisted of two distinct components: spike-wave (SW) or polyspike-wave (PSW) at 2-3 Hz and fast runs at 10-15 Hz. Because the thalamus is an input source and target of cortical neurons, we investigated here the seizure behavior of thalamic reticular (RE) and thalamocortical (TC) neurons, two major cellular classes that have often been implicated in the generation of paroxysmal episodes. We performed single and dual simultaneous intracellular recordings, in conjunction with multisite field potential and extracellular unit recordings, from neocortical areas and RE and/or dorsal thalamic nuclei under ketamine-xylazine and barbiturate anesthesia. Both components of seizures were analyzed, but emphasis was placed on the fast runs because of their recent investigation at the cellular level. 1) The fast runs occurred at slightly different frequencies and, therefore, were asynchronous in various cortical neuronal pools. Consequently, dorsal thalamic nuclei, although receiving convergent inputs from different neocortical areas involved in seizure, did not express strongly synchronized fast runs. 2) Both RE and TC cells were hyperpolarized during seizure episodes with SW/PSW complexes and relatively depolarized during the fast runs. As known, hyperpolarization of thalamic neurons deinactivates a low-threshold conductance that generates high-frequency spike bursts. Accordingly, RE neurons discharged prolonged high-frequency spike bursts in close time relation with the spiky component of cortical SW/PSW complexes, whereas they fired single action potentials, spike doublets, or triplets during the fast runs. In TC cells, the cortical fast runs were reflected as excitatory postsynaptic potentials appearing after short latencies that were compatible with monosynaptic activation through corticothalamic pathways. 3) The above data suggested the cortical origin of these seizures. To further test this hypothesis, we performed experiments on completely isolated cortical slabs from suprasylvian areas 5 or 7 and demonstrated that electrical stimulation within the slab induces seizures with fast runs and SW/PSW complexes, virtually identical to those elicited in intact-brain animals. The conclusion of all papers in this series is that complex seizure patterns, resembling those described at the electroencephalogram level in different forms of clinical seizures with SW/PSW complexes and, particularly, in the Lennox-Gastaut syndrome of humans, are generated in neocortex. Thalamic neurons reflect cortical events as a function of membrane potential in RE/TC cells and degree of synchronization in cortical neuronal networks.

Adrenergic alpha-Agonists↗

Twelve-hour phase shifts of hamster circadian rhythms elicited by voluntary wheel running.

Running in a novel wheel can serve as a nonphotic zeitgeber to entrain or phase shift circadian rhythms in hamsters. In this study, hamsters were entrained to a light:dark schedule of 14:10 h but had no access to running wheels. At four different phase points of the light cycle, hamsters were transferred to constant darkness and provided with running wheels. All hamsters began running shortly after transfer and were allowed to continue running at their own volition. Approximately 20% of the hamsters transferred at zeitgeber time (ZT) 23 (ZT 12 = lights out) ran more than 4 h after transfer and showed phase advances of the circadian activity rhythm by as much as 15 h, while hamsters that ran less than 4 h on average did not phase shift. A similar result was observed for hamsters transferred at ZT 2. Hamsters transferred at ZT 5 and 8 also did not phase shift if they ran less than 4 h, although the relation between longer runs and phase shifts became less evident. A sustained run in excess of 4 h appeared to be associated with large phase advances. These results show that under certain conditions, a single sustained bout of wheel-running activity is capable of phase shifting the circadian pacemaker by more than 12 h.

Animals↗

Locking and unlocking of running wheel affects circadian period stability differently in three inbred strains of rats.

Running-wheel access has been shown to shorten the circadian period length (tau) of various mammalian species. Due to the close correlation between tau and the level of activity, running wheel-induced changes of the activity level are thought to be responsible for the observed changes in tau. In the present study, the influence of the running wheel on tau and the activity level was examined in three inbred strains of rats (ACI, BH, LEW). Four animals of each strain had free access to their running wheels, while the wheels of the other 4 animals of each strain were mechanically locked. These conditions were changed twice, so that each animal encountered both kinds of changes, that is, from a locked to an unlocked running wheel and vice versa. During the whole study, overall activity was measured by infrared detectors. Running-wheel access resulted in a significant increase of overall activity in strains LEW and ACI. However, significant changes of tau were observed only in LEW rats. These rats showed a significant shortening of tau after the second change of the housing conditions regardless of whether the wheel was locked or unlocked. Consequently, no causal relationship was found between changes of tau and running wheel-induced changes of overall activity. Instead, the results suggest that subtle environmental influences like locking or unlocking the running wheel affect tau in a strain-dependent manner, whereas changes in the activity level are neither necessary nor sufficient to induce changes of tau.

Animals↗

Decreased wheel-running activity in hamsters post myocardial infarction.

Reduced exercise capacity is a key symptom and an independent determinant of mortality in patients with heart failure. We analyzed the running activity of hamsters with cardiac dysfunction after myocardial infarction. In 39 male Syrian hamsters aged 10 to 12 weeks, a myocardial infarction (MI) was produced by permanent ligation of the left coronary artery. Spontaneous running activity in a wheel was monitored daily. After four weeks, left ventricular (LV) hemodynamics (catheter tip manometry) were measured at baseline and during inotropic stimulation (isoprenaline 0.03, 0.1 and 0.3 microg/kg/min i.v.). LV infarct size was quantified using planimetry. Four weeks post MI, daily running distance was reduced stepwise in animals with small (4-15% of LV: 9.8 +/- 3.4 km/d) and large (> 15% of LV: 7.5 +/- 3.5 km/d) MI, compared to sham-operated hamsters (11.5 +/- 1.5 km/d). Similar reductions were observed in maximum speed and distance of longest running period. MI size influenced daily running distance, maximum speed, and longest running period (linear correlations, all p < 0.05). MI size also impaired LV systolic and diastolic function under isoprenaline stimulation. The results suggest that myocardial infarction reduces running capacity and isoprenaline stimulated LV function in hamsters, mimicking impaired exercise performance in patients with heart failure. Analysis of running activity in hamsters with myocardial infarction offers a unique opportunity for non-invasive and serial functional assessment of heart failure in the experimental setting.

Journal Article↗

Running wheel choice by Syrian hamsters.

The present study investigated the preference of male and female Syrian hamsters, Mesocricetus auratus, for different types of running wheels. Hamsters were placed individually in sets of multiple cages linked by tunnels, each cage with a different running wheel. The number of wheel revolutions in each cage was tallied daily over 40 days. The hamsters did not express a preference when offered a choice of a running surface made of metal rods spaced 9 mm apart and a similar running surface covered in plastic mesh to prevent the possible slippage of feet between the rods. The hamsters did express a clear preference for larger wheels (35 versus 23 cm diameter), and for completely circular wheels over truncated ones. They neither favoured nor rejected wheels with small obstacles along the running surface. In all experiments, preferences were more strongly expressed by males than by females. Running wheels for hamsters may be improved by enlarging their diameter (to the standards often used for rats, if practically possible) and by ensuring good footing on the running surface (a space no larger than 9 mm between evenly spaced rods seems sufficient to achieve this, at least in large wheels and for hamsters older than 55 days). Installing obstacles along the running surface does not appear to make the wheel more interesting to hamsters.

Animals↗

[Experimental study on the effect of forced running on occurrence of osteoarthritis in the knee of C 57 BL mice].

We investigated the effects of running load on the development of osteoarthritis (OA) in C 57 black male mice (Silberberg), a strain which spontaneously develops OA of the knee joint, and in Std:ddy male mice, a strain which does not develop OA. Six-week-old mice of both strains were randomly assigned to a non-forced running (control) group or forced running groups, which ran on treadmill 1 or 2 hours per day of 5 day/week program. Six, 18, and 30 weeks after the running period, the mice were sacrificed and the knee joints examined histologically and bone morphometrically. In the control C 57 black mice, OA occurred at 6 months of age, while in the forced running C 57 black mice, OA occurred at 3 months of age (6 weeks after the end forced running). In 6 and 9 months old C 57 black mice the incidence of OA in the forced running group was higher than that in corresponding control group. These data suggest that forced running load accelerates the process of OA in the knee joint in young mice of a strain which spontaneously develops OA.

Animals↗

[Treadmill running enhances the ability of learning in young rats.].

To investigate the effect of treadmill running on the ability of learning in young rats, male Sprague-Dawley rats (5 weeks of age) were used for the experiment. Animals were randomly divided into the control and running groups (n=15 in each group). The rats in running group were made run on a motor-driven treadmill for 1 week at a speed of 2 m/min for the first 5 min, at a speed of 5 m/min for the next 5 min, then at a speed of 8 m/min for the last 20 min. Then the Morris water maze was used to observe learning and memory ability of rats in both groups. The tests consisted of place navigation and spatial probe test. We found that, in place navigation training, the latency of rats in running group was less than that in control group (P<0.05); and from the third training session on, there was significant difference between the rats in control and running groups in swimming velocity (P<0.01); furthermore, it was observed that the rats in running group had stronger motivation and more exact orientation in searching for platform, which could be indicated by the index of turn angle and angular velocity. In spatial probe test, there was no significant difference between the two groups in swimming velocity, percentage of swimming distance and frequency of crossing platform in D quadrant, where the platform situated (P>0.05). These findings suggest that low speed treadmill running can enhance the ability of learning in young rats.

Age Factors↗

The between-run component of variation in internal quality control.

Design of control charts for the mean, the within-run component of variance, and the ratio of between-run to within-run components of variance is outlined. The between-run component of variation is the main source of imprecision for analytes determined by an enzymo- or radioimmunoassay principle; accordingly, explicit control of this component is especially relevant for these types of analytes. Power curves for typical situations are presented. I also show that a between-run component of variation puts an upper limit on the achievable power towards systematic errors. Therefore, when the between-run component of variation exceeds the within-run component, use of no more than about four controls per run is reasonable at a given concentration.

Analysis of Variance↗

Plastid run-on transcription. Application to determine the transcriptional regulation of spinach plastid genes.

We have developed a spinach plastid run-on transcription system to determine the extent of transcriptional regulation of chloroplast genes during morphogenetic changes of the organelle. In contrast to transcription in a spinach chloroplast extract, which requires initiation of exogenously added genes (Gruissem, W., Greenberg, B. M., Zurawski, G., and Hallick, R. B. (1983) Cell 35, 815-828), RNA synthesis in the run-on system is not affected by heparin or different salt concentrations. Transcription is asymmetric, and the size of the run-on transcripts varies between 75 nucleotides and 8 kilobases. Quantitative filter hybridization studies included gene-specific probes for the ribosomal RNA genes and nine protein-coding genes. Based on the amounts of hybridizable run-on transcripts, these genes can be ordered according to their respective transcriptional activities. The relative transcriptional activities of psbA, rbcL, and atpB in the run-on assay correlate closely with their reported promoter strengths in vitro. The plastid run-on transcription assay has been applied to determine the transcriptional regulation of plastid genes. Hybridization of run-on transcripts to regions of the spinach chloroplast genome containing at least nine tRNA genes indicates that most or all loci are highly transcribed. No significant qualitative and quantitative differences are detected when run-on transcripts from plastids of etiolated and greening cotyledons are hybridized to total, restriction enzyme-digested chloroplast DNA, demonstrating limited transcriptional regulation during chloroplast development.

Chloroplasts↗

The prospects of daytime running lights for reducing vehicle crashes in the United States.

Daytime running lights increase visual contrast between vehicles and their background, improving their noticeability and detectability. Seven countries require motor vehicles to have lights on during all daytime periods--Canada, Denmark, Finland, Hungary, Iceland, Norway, and Sweden. Studies from these and other countries have generally indicated that daytime running lights use is associated with small to moderate reductions in multiple-vehicle daytime crashes, especially those involving vehicles approaching from the front or side. There is evidence also that initial positive effects of daytime running lights do not dissipate over time, that is, there is little support for novelty or habituation effects. The bulk of the evidence suggests that running lights do not lead to increases in collisions involving pedestrians and pedalcyclists, allaying concerns that there would be negative consequences of making these road users relatively less conspicuous. Most of the studies have been conducted in countries located at latitudes that are to the north of most of the continental United States and that have longer twilight periods and generally lower ambient illumination. The concern has been expressed that running lights may lose their effectiveness in countries located at lower latitudes, such as the United States, because the lights will provide less of a contrast. General Motors Corporation and some other manufacturers are now providing running lights on new models with higher intensities than are used in Scandinavian countries. Findings in running lights studies suggest that their effect in the United States will be positive, and their introduction provides an opportunity to determine the effect. The costs of running lights are low, so even modest crash reductions would be cost effective.

Accidents, Traffic↗

One-mile run performance and cardiovascular fitness in children.

BACKGROUND: Endurance run tests are administered in schools to assess cardiovascular fitness, defined in the laboratory as maximum oxygen consumption. OBJECTIVE: To examine the validity of this concept, assessing the influences of body fat and maximum values of oxygen consumption per unit time, stroke volume, heart rate, and arteriovenous oxygen difference on 1-mile (1.6-km) run time in healthy sixth-grade boys. STUDY DESIGN: Subjects were 36 boys with a mean (SD) age of 12.2 (0.5) years. The relationship was examined between body fat content (estimated by skinfold measurements) and maximum oxygen consumption per kilogram and cardiac variables (during maximum cycle testing) with 1-mile run velocity. RESULTS: Body fat content and maximum oxygen consumption per kilogram (independent of body fat) accounted for 31% and 28% of the variance in run velocity, respectively. Stroke volume was the only component of maximum oxygen consumption that related to run performance. CONCLUSIONS: These findings suggest that cardiovascular fitness and body fat content contribute equally to 1-mile run time in healthy boys and together account for only 60% of the variance in performance on this endurance fitness test. Consequently, 1-mile run performance in children may not serve as a strong indicator of cardiovascular fitness.

Adipose Tissue↗