Identification of four families of yCCR4- and Mg(2+)-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg(2+)-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals

Identification of retinoic acid-regulated nuclear matrix-associated protein as a novel regulator of gastric cancer

Background: Retinoic acid-regulated nuclear matrix-associated protein (RAMP) is a WD40 repeat-containing protein that is involved in various biological functions, but little is known about its role in human cancer. This study aims to delineate the oncogenic role of RAMP in gastric carcinogenesis

Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells

CARDIORESPIRATORY RESPONSES TO THE USFS WILDLAND FIREFIGHTER ARDUOUS PACK TEST

C.J. Alfiero,C.L. Dumke FACSM, B.C. Ruby FACSM, M.W. Bundle University of Montana, Missoula, MT US wildland firefighters administer over 30,000 physical tests per year to qualify candidates for the occupational demands of fire suppression. The primary assessment is the arduous pack test (APT) a 4.83 km hike that must be completed in 45 min while wearing a 20.45 kg pack. Delivery of individual feedback to guide the physical training of candidates is hampered by two factors; first, passing the pack test is widely considered the minimum performance level necessary needed for this occupation, and second, the binary nature of the assessment presents candidates with a task representing an unknown and self-selected exercise intensity. PURPOSE: To determine the cardiorespiratory response elicited by the APT within a subject population whose aerobic capacity and body masses vary. METHODS: 61 young (age = 22.8 ± 3.2 yrs) adults (36 males, Mb = 79.5 ± 8.8kg; 25 females, Mb = 67.5 ± 13.5kg; study range: 55.4 - 119.6 kg) performed the APT and subsequently underwent a hiking inclined-treadmill test to VO2peakwhile wearing a skin mounted heart rate (HR) monitor and 20.45-kg pack. RESULTS: 50 of the 63 subjects achieved the 45-min cutoff with a finishing time of 41.8 ± 2.1min, the non-passers had a mean time of 47.7 ± 2.7 min. Non-passers were 77% female and 23% male. The VO2peakvalues of the passing and non-passing groups were 49.4 ± 7.2 and 42.6 ± 9.6 mlO2kg-1 min-1, respectively; the study range was 62.1 to 30.8 mlO2kg-1 min-1. HR, whether expressed as a fraction of the subject’s maximum rate (passers = 81.2 ± 17.1 and non-passers = 79.9 ± 12.7% of HRmax), or as the fraction of the HR reserve (passers = 68.0 ± 7.9 and non-passers = 67.7 ± 15.3% of HR reserve) were not different between the groups. Regression of VO2peakon completion time yielded a negative relationship (R2 = 0.45). In contrast, the HR responses and completion time were consistent among the participants (R2\u3c 0.01 for both % of HRmaxand % of HR reserve). CONCLUSION: To successfully complete the APT candidates must achieve a HR reserve of 68% or less while maintaining a walking speed of 1.8 m s-1. These data suggest that monitoring HR during load carriage may be used to identify candidates with adequate and inadequate pre-fire season readiness

THE IMPACT OF HYDRATION STATUS DURING HEAT ACCLIMATION ON PHYSIOLOGICAL STRAIN AND EXERCISE PERFORMANCE

M.W. Schleh, M. Johannsen, B.C. Ruby FACSM, C.L. Dumke FACSM The University of Montana, Missoula, MT Heat acclimation improves thermal tolerance and attenuates the physiological and perceptual strain associated with exercise in the heat. A limited fluid delivery strategy during heat acclimation may promote further adaptations to attenuate heat stress over long periods, decreasing the probability of heat related illness. PURPOSE: To determine i) the effect of fluid delivery by inducing dehydration (DEH=0.5 ml*kg-1*min-15) compared to euhydration (EUH=2.0 ml * kg-1 * 15 min-1) following three heat acclimation bouts on heat stress factors, and ii) to determine the effect of aerobic performance in the heat following DEH and EUH acclimation trials. METHODS: Eight aerobically fit males (23.0±0.9 yrs, 82.1 ± 2.0 kg, 184.0 ± 2.5 cm, 53.2 ± 1.5 ml*kg-1*min-1 VO2 max) completed 90-minute heat stress test (HST) in hot conditions (T=40°C, RH=30%) at 50% VO2 max prior to and following three-days of EUH and DEH acclimation trials. Acclimation trials consisted of three heat exposures in the same environmental conditions, intensity, and duration as previously stated on alternating days. Participants wore standard wildland firefighter (WLFF) Nomex: yellow shirt, green pants, and a cotton T-shirt. Following each HST, aerobic performance was assessed by a graded ramp protocol by increasing treadmill grade by 1% until 15%, and then increased 1 mph every minute until volitional exhaustion. Core temperature (Tc) and Skin temperature (Tsk) were measured continuously, and reported as peak TC and TSk at 90 minutes. Sweat rate by ∆nude weight, and post acclimation performance measured in time to exhaustion. All data was analyzed by a two-way analysis of variance (ANOVA) 2 (trt) x 2 (time). RESULTS: Peak Tc and Tsk during HST significantly decreased in effect for time in DEH & EUH groups (p\u3c0.001, p=0.003) respectively. Sweat rate was significantly greater in DEH compared to EUH (trt*time: p=0.034). Aerobic performance increased greater in EUH compared to DEH (trt*time: p= 0.034). CONCLUSION: Short-term heat acclimation is an effective strategy to attenuate physiological strain associated with heat stress, and also improve aerobic performance in the heat. Fluid delivery strategies during acclimation do not affect thermal strain, but may increase sweat capacity. Controlled dehydration during acclimation did not however increase performance. This project was supported by the US Forest Service

EFFECT OF HIGH INTENSITY INTERVAL TRAINING ON MAXIMAL FATTY ACID OXIDATION VERSUS STEADY STATE EXERCISE

K. Beisel, E. Donahue, A. Kalafatis, K. Christison, C.L. Dumke, FACSM University of Montana, Missoula, MT Previous evidence suggests high intensity intervals and steady state exercise can enhance fat oxidation. These two stimuli have not been examined in the same exercise bout. PURPOSE: This study examines the effect of high intensity interval training (HIIT) on fatty acid oxidation compared to steady state at each individuals maximal fatty acid oxidation (MFAO). METHODS: Twelve fasted, uncaffeinated individuals (8 males, 4 females, average VO2max = 44.3±2.3 ml·kg-1·min-1, average age =23.8±0.8 years) underwent a randomized crossover design study with 2 trials; trial A included cycling for 60 minutes at MFAO workload (0.55±0.05 g·min-1; 58.6±1.1 % VO2max; 126.7±9.6 W) and trial B included 3 minute intervals at 90% and 30% of maximal workload during the first 30 minutes, and the last 30 minutes the subjects cycled at the MFAO workload. Total work was matched between the two trials. Heart rate (HR), respiratory exchange ratio (RER), rating of perceived exertion (RPE), absolute fat (FAO) and carbohydrate oxidation (CHOO) were recorded every 15 minutes, lactate and glucose taken every 30 minutes. Data was analyzed using a 2X2 repeated measures ANOVA and paired samples T-test for overall trial differences. RESULTS: There was a main effect of time on lactate (p=0.002), RPE (p=0.027), HR (45 min: 164.6±2.8 bpm vs. 60 min: 168.0±2.9 bpm, 45 min: 159.4±3.6 bpm vs. 60 min:159.8±4.6 bpm, p=0.028), and FAO (45 min: 0.55±0.05 g/min vs. 60 min: 0.66±0.06 g/min, 45 min: 0.62±0.04 g/min vs. 60 min: 0.63±0.04 g/min, p=0.044). There was a trial effect of RPE (45 min: 13.5±0.3 vs 60 min: 13.8±0.4, 45 min: 11.4±0.8 vs 60 min: 12.5±0.6, p =0.014), and sweat rate (0.91±0.07 L·min-1 vs 0.69±0.03 L·min-1, p=0.03) All listed values are trial A vs. trial B respectively. A trial by time interaction was found for lactate (pCONCLUSION:These data suggest as exercise endurance increases, MFAO is elevated. Despite elevated lactate in the HIIT trial FAO did not differ between trials. Despite matched work between trials, significant reductions in RPE, HR and SR, suggests subjects had reduced metabolic stress at MFAO workloads following HIIT

MAXIMAL FAT OXIDATION DURING STEADY STATE EXERCISE

A. Kalafatis, K. Beisel, E. Donahue, K. Christison, C.L. Dumke, FACSM University of Montana, Missoula, MT Research has demonstrated multiple factors contributing to maximal fatty acid oxidation (MFAO). However, less is known about factors that regulate changes in fatty acid oxidation during exercise. PURPOSE: The purpose of this study is better understand how cycling at MFAO for 60 minutes will alter fat oxidation. METHODS: Twelve fasted, uncaffeinated individuals (8 males, 4 females, average VO2max = 44.3± 2.3 mL·kg-1·min-1, average age =23.7±0.8) underwent two laboratory visits. They first underwent a VO2 max test in which heart rate (HR), rating of perceived exhaustion (RPE), absolute fat (FAO) and carbohydrate oxidation (CHOO) were recorded. Hydrostatic weighing was used to determine body fat percentage. The second visit included a 60-minute exercise on a cycle ergometer at the subjects predetermined maximal fatty acid oxidation (MFAO) workload (0.55±0.05 g·min-1; 58.6±1.1 % VO2max; 126.7±9.6 W). HR, RPE, and the respiratory exchange ratio were recorded every fifteen minutes, with water provided at 3 ml·kg-1 at 15, 30, and 45 minutes. Lactate and glucose were measured at 30 and 60 minutes. Data were analyzed using repeated measures ANOVA, and paired samples T-test for pre- and post changes. RESULTS: There was a main effect of time on HR (154.7±3.5 at 15min, vs 168.0±2.9 bpm at 60min; p=0.005), RPE (11.2±0.3 at 15 min vs 13.8±0.4 at 60 min; p=0.001) and FAO (0.46±0.05 at 15 min vs. 0.65±0.06 at 60 min; p\u3c0.001). FAO at the end of 60 mins of exercise was significantly greater than MFAO (p=0.04) from the VO2max test. The change in FAO during exercise did not correlate with subject characteristics. However, MFAO was superior in the tallest (r=0.67, p=0.02), heaviest (r=0.78, p=0.003), and those with the greatest maximum workload (r=0.82, p=0.001). Correlation was found between the tallest and weight loss during the trial (r=0.66, p=0.019). CONCLUSION: These data suggest that during the 60 minute bike ride that FAO exceeds the MFAO during a VO2max test. In addition, the largest subjects with the greatest maximum workload had the highest MFAO and FAO during the one hour trials

THE EFFECT OF DEHYDRATION ON HEAT ACCLIMATION

R. Berland, C. Briscoe, M. Schleh, B.C. Ruby FACSM, C.L. Dumke FACSM The University of Montana, Missoula, MT With the prevalence of occupations occurring in hot environments, it is crucial to develop a protocol to protect these individuals from heat related illness. We hypothesize that heat acclimation with mitigated fluid delivery will improve cardiovascular and perceptual strain associated with heat stress, and hydration maintenance. PURPOSE: To determine the impact of hydration status during heat acclimation on hydration retention and cardiovascular and perceptual drift. METHODS: Eight aerobically fit male subjects (23 ± 1.00 yrs, 82.1±2.14 kg, 53.18 ± 1.63 mL•kg-1•min-1 VO2 max, 13.8% ± 1.31% BF) completed a heat stress test (HST) two days prior to and following three acclimation bouts, in alternating days to allow for rehydration. HST and acclimation bouts consisted of 90 min treadmill walking at 50% VO2 max in hot conditions (40℃, 30% RH). Fluid delivery during acclimation bouts was administered in a randomized crossover design, where fluids were administered to promote a dehydrated (DEH = 0.5 mL/kg) or a euhydrated (EUH = 2.0 mL/kg) state. A five week washout period separated DEH from EUH acclimation periods. Participants wore standard wildland firefighter (WLFF) NoMex clothing with a cotton shirt. Peak heart rate (HR), peak rate of perceived exertion (RPE), percent dehydration (%DEH), and ΔBW were analyzed by a 2(trt) X 2(trial) ANOVA. RESULTS: HR decreased from PRE to POST (p\u3c0.001) (178.9±13.3, 165.1±16.0, 182.3±11.8, 162.9±16.0, for DEH vs EUH pre-post respectively) but there was not an effect of treatments. RPE decreased between trials (p\u3c0.001)(17.8±2.3, 14.5±2.6, 16.9±2.5, 13.6±2.3 for DEH vs EUH pre-post respectively) but no significance between treatments. ΔBW demonstrated a time and trtXtime interaction (p=0.030) with greater ΔBW in the DEH trial following acclimation (1.15±0.27, 1.83±0.39, 1.35±0.35, 1.57±0.38 for DEH vs EUH pre-post respectively). %DEH demonstrated a time and trtXtime interaction (p=0.033) with greater %DEH in the DEH trial following acclimation (1.48±0.38, 2.15±0.48, 1.72±0.49, 1.99±0.52 for DEH vs EUH pre-post respectively). CONCLUSION: Our data suggests that heat acclimation reduces cardiovascular and perceptual strain, however mitigated fluid delivery during acclimation did not alter this adaptation. Mitigated fluid delivery increases ΔBW and %DEH suggesting greater sweat capacity following the acclimation trials. This suggests that controlled dehydration may result in greater adaptations during short term heat acclimation. Funding provided by the US Forest Service