Robust neurite extension following exogenous electrical stimulation within single walled carbon nanotube-composite hydrogels.

UNLABELLED: The use of exogenous electrical stimulation to promote nerve regeneration has achieved only limited success. Conditions impeding optimized outgrowth may arise from inadequate stimulus presentation due to differences in injury geometry or signal attenuation. Implantation of an electrically-conductive biomaterial may mitigate this attenuation and provide a more reproducible signal. In this study, a conductive nanofiller (single-walled carbon nanotubes [SWCNT]) was selected as one possible material to manipulate the bulk electrical properties of a collagen type I-10% Matrigel™ composite hydrogel. Neurite outgrowth within hydrogels (SWCNT or nanofiller-free controls) was characterized to determine if: (1) nanofillers influence neurite extension and (2) electrical stimulation of the nanofiller composite hydrogel enhances neurite outgrowth. Increased SWCNT loading (10-100-µg/mL) resulted in greater bulk conductivity (up to 1.7-fold) with no significant changes to elastic modulus. Neurite outgrowth increased 3.3-fold in 20-µg/mL SWCNT loaded biomaterials relative to the nanofiller-free control. Electrical stimulation promoted greater outgrowth (2.9-fold) within SWCNT-free control. The concurrent presentation of electrical stimulation and SWCNT-loaded biomaterials resulted in a 7.0-fold increase in outgrowth relative to the unstimulated, nanofiller-free controls. Local glia residing within the DRG likely contribute, in part, to the observed increases in outgrowth; but it is unknown which specific nanofiller properties influence neurite extension. Characterization of neuronal behavior in model systems, such as those described here, will aid the rational development of biomaterials as well as the appropriate delivery of electrical stimuli to support nerve repair. STATEMENT OF SIGNIFICANCE: Novel biomedical devices delivering electrical stimulation are being developed to mitigate symptoms of Parkinson's, treat drug-resistant depression, control movement or enhance verve regeneration. Carbon nanotubes and other novel materials are being explored for novel nano-neuro devices based on their unique properties. Neuronal growth on carbon nanotubes has been studied in 2D since the early 2000s demonstrating increased outgrowth, synapse formation and network activity. In this work, single-walled carbon nanotubes were selected as one possible electrically-conductive material, dispersed within a 3D hydrogel containing primary neurons; extending previous 2D work to 3D to evaluate outgrowth within nanomaterial composites with electrical stimulation. This is the first study to our knowledge that stimulates neurons in 3D composite nanomaterial-laden hydrogels. Examination of electrically conductive biomaterials may serve to promote regrowth following injury or in long term stimulation.

Effect of ultra-endurance exercise on left ventricular performance and plasma cytokines in healthy trained men.

The purpose of this study was to investigate the effect of ultra-endurance exercise on left ventricular (LV) performance and plasma concentration of interleukin (IL)-6, IL-10, IL-18 and tumour necrosis factor alpha (TNF-α) as well as to examine the relationships between exercise-induced changes in plasma cytokines and those in echocardiographic indices of LV function in ultra-marathon runners. Nine healthy trained men (mean age 30±1.0 years) participated in a 100-km ultra-marathon. Heart rate, blood pressure, ejection fraction (EF), fractional shortening (FS), ratio of early (E) to late (A) mitral inflow peak velocities (E/A), ratio of early (E') to late (A') diastolic mitral annulus peak velocities (E'/A') and E-wave deceleration time (DT) were obtained by echocardiography before, immediately after and in the 90th minute of the recovery period. Blood samples were taken before each echocardiographic evaluation. The ultra-endurance exercise caused significant increases in plasma IL-6, IL-10, IL-18 and TNF-α. Echocardiography revealed significant decreases in both E and the E/A ratio immediately after exercise, without any significant changes in EF, FS, DT or the E/E' ratio. At the 90th minute of the recovery period, plasma TNF-α and the E/A ratio did not differ significantly from the pre-exercise values, whereas FS was significantly lower than before and immediately after exercise. The increases in plasma TNF-α correlated with changes in FS (r=0.73) and DT (r=-0.73). It is concluded that ultra-endurance exercise causes alterations in LV diastolic function. The present data suggest that TNF-α might be involved in this effect.

Cardiovascular and hormonal responses to static handgrip in young and older healthy men.

The purpose of this study was to investigate the effect of age on cardiovascular changes and plasma concentrations of adrenomedullin (ADM), catecholamines, endothelin-1 (ET-1) and plasma renin activity (PRA) in healthy men. A total of 15 young (21 ± 0.3 years) and 15 older (64 ± 0.7 years) healthy men performed two 3-min bouts of static handgrip at 30% of maximal voluntary contraction, alternately with each hand without any break between the bouts. During exercise heart rate (HR), blood pressure (BP), stroke volume (SV) and pre-ejection period (PEP) and left ventricle ejection time (LVET) were measured. Blood samples were taken before exercise, at the end of both exercise bouts and in the fifth minute of the recovery period. The handgrip-induced increases in HR and cardiac output were significantly smaller in older than in young men (p < 0.01). SV decreased only in older men (p < 0.001). There were no differences between groups in BP increases. The baseline plasma ADM and catecholamines were higher in older man compared to young subjects. Handgrip caused increases in plasma ADM, ET-1 and PRA only in older men (p < 0.05). The increases in plasma ADM correlated positively with those of noradrenaline (NA), PRA, ET-1 and LVET and negatively with changes in total peripheral resistance (TPR), SV, PEP and PEP/LVET ratio. The increases in plasma ET-1 correlated positively with those of NA, PRA, TPR, mean BP and SV. These results revealed that ADM, ET-1 and angiotensin II can contribute to maintain vascular tone during static exercise in older but not in younger men.

Metabolic and hormonal responses to body carbohydrate store depletion followed by high or low carbohydrate meal in sedentary and physically active subjects.

The study was designed to determine metabolic and hormonal responses to acute modification of body carbohydrate stores by exercise and subsequent meals and to find out whether the responses depend on the training status of subjects. Nine sedentary students and 10 endurance athletes took part in four experimental sessions. During control session, after overnight fast oxygen uptake and CO2 production were measured and blood glucose, free fatty acids (FFA), insulin (I), leptin (L), growth hormone (GH), testosterone (T), catecholamines, ACTH and cortisol were determined. The remaining sessions were preceded by 1.5 h exercise at 70% HRmax in the evening followed by 12-16 hrs fast till morning when subjects ate either high-carbohydrate (H-CHO) or low-carbohydrate (L-CHO) meal or fasted. Respiratory gases and blood samples were collected before and 2 hours after meal. In glycogen depleted subjects respiratory quotient (RQ), I, norepinephrine (NE) and L decreased, whilst other variables were unaltered. Changes in I and NE were greater in athletes than in sedentary subjects. After H-CHO RQ, blood glucose, I and NE increased and FFA, GH and T decreased. The latter effect was greater in athletes than in untrained subjects. After L-CHO, RQ was at the fasting level and FFA increased only in sedentary group. In both groups I increased and GH and T decreased. Neither meal affected L concentration. In conclusion, hormonal and metabolic changes observed after depleting carbohydrate stores resemble those occurring during starvation. Composition of the ingested meal affects postprandial metabolism, which additionally depends on the subjects' training status.

Integration of the thiol redox status with cytokine response to physical training in professional basketball players.

The present study was designed to evaluate the plasma markers of reactive oxygen species (ROS) activity and cytokines, and their relationship with thiol redox status of basketball players during training. Sixteen professional players of the Polish Basketball Extraleague participated in the study. The study was performed during the preparatory period and the play-off round. Markers of ROS activity (lipid peroxidation TBARS, protein carbonylation PC) and reduced glutathione (GSH) demonstrated regularity over time, i.e. TBARS, PC and GSH were elevated at the beginning and decreased at the end of training periods. Oxidized glutathione (GSSG) was not affected by exercise training. Thiol redox status (GSH(total)-2GSSG/GSSG) correlated with TBARS and PC in both training periods. The level of interleukin-6 (IL-6) was increased and positively correlated with thiol redox (r=0.423) in the preparatory period, whereas tumor necrosis factor alpha (TNFalpha) was increased and inversely correlated with thiol redox (r= 0.509) in the play-off round. The present study showed significant shifts in markers of ROS activity, thiol redox status and inflammatory mediators (IL-6, TNFalpha) following professional sport training as well as correlation between changes in thiol redox and cytokine response.

Influence of body carbohydrate store modification on catecholamine and lactate responses to graded exercise in sedentary and physically active subjects.

Aim of this study was to elucidate the influence of carbohydrate store modification on exercise capacity and catecholamine thresholds. Nine sedentary students and 10 endurance athletes volunteered for the study consisting of four sessions in one-week intervals. During control session (C) subjects performed graded exercise till exhaustion with workload increasing by 50 Watts every 3 min after an overnight fast. Blood lactate and catecholamines were determined at rest, after each workload and at exhaustion. In the evening preceding each of the remaining three sessions subjects performed glycogen reducing exercise lasting 1.5 hrs at 70% HRmax. Till next morning they did not eat any meal but drank water. Two hours before exercise subjects were given either a high-carbohydrate (H-CHO) or a low-carbohydrate (L-CHO) meal of approx. 1000 kcal or remained fasted (F). Depletion of carbohydrate stores enhanced contribution of fat-derived energy substrates at submaximal workloads, but did not influence either maximal oxygen consumption, workload and lactate concentration or lactate threshold. Low carbohydrate availability resulted in elevated concentration of catecholamines only in untrained subjects. Ingestion of a meal either high or low in carbohydrates diminished those changes. Catecholamine thresholds were similar in all sessions and higher in athletes' group.

Effect of 6-week endurance training on hemodynamic and neurohormonal responses to lower body negative pressure (LBNP) in healthy young men.

Endurance training is considered as a factor impairing orthostatic tolerance although an improvement and lack of effect have been also reported. The mechanisms of the changes and their relation to initial tolerance of orthostasis are not clear. In the present study, effect of moderate running training on hemodynamic and neurohormonal changes during LBNP, a laboratory test simulating orthostasis, was investigated in subjects with high (HT) and low (LT) tolerance of LBNP. Twenty four male, healthy subjects were submitted to graded LBNP (-15, -30 and -50 mmHg) before and after training. During each test heart rate (HR), stroke volume (SV) and blood pressure, plasma catecholamines, ACTH, adrenomedullin, atrial natriuretic peptide, and renin activity were determined. Basing on initial test, 13 subjects who withstood LBNP at -50 mmHg for 10 min were allocated into HT group and 11 subjects who earlier showed presyncopal symptoms to LT group. Training improved LBNP tolerance in six LT subjects. This was associated with attenuated rate of HR increase and SV decline (before training, at -30 mmHg deltaHR was 21 +/- 4 beats/min and deltaSV - -36+/- 8 ml while after training the respective values were 8 +/- 4 beats/min and -11+/- 6 ml). No differences in hemodynamic response were found in HT subjects and those from LT group whose LBNP tolerance was unchanged. In neither group training affected neurohormonal changes except inhibition of plasma ACTH rise in subjects with improvement of LBNP tolerance. It is concluded that some subjects with low orthostatic tolerance may benefit from moderate training due to improvement of cardiac function regulation.

Cardiovascular and neurohormonal responses to lower body negative pressure (LBNP): effect of training and 3 day bed rest.

Both intensive training and bed confinement impair orthostatic tolerance, however, moderate training may exert beneficial effect on cardiovascular adjustment to gravitational stimuli. It was hypothesized that moderate training attenuates effects of bed rest. To test this assumption 24 healthy male volunteers aged 20.8+/-0.9 yrs were subjected to 6 degrees head down bed rest (HDBR) for 3 days before and after 6 weeks of moderate endurance training. Before and after HDBR graded LBNP tests (-15, -30, -50 mmHg) were performed. During these tests heart rate (HR), stroke volume (SV), blood pressure (BP), plasma catecholamines, ACTH, adrenomedullin, atrial natriuretic peptide, plasma renin activity (PRA) and hematocrit were determined. HDBR did not systematically influence LBNP tolerance up to -50 mmHg, but it enhanced rates of reduction of SV, cardiac output and systolic BP and increased elevations of HR and PRA. Training did not alter significantly effects of HDBR on LBNP-induced changes in HR, SV, CO and TPR but it attenuated decrease in systolic BP and diminished increases in plasma noradrenaline and PRA. In conclusion, training has negligible effect on the HDBR-induced changes in central hemodynamics during LBNP but may increase vascular sensitivity to some vasoconstricting factors.

Early effects of short-term endurance training on hormonal responses to graded exercise.

Twelve male, sedentary volunteers (22.0 +/-) were submitted to three weeks of a bicycle ergometer training, consisting of 45 min exercise (at 70% VO2max), 4 times in the first week and 3 times in the next 2 weeks. They performed four incremental exercise tests with the power output increased by 50 W every 3 min until volitional exhaustion: two before training (C1 and C2), and after one (T1) and three (T3) weeks of training. Before and after each load the plasma noradrenaline (NA), adrenaline (A) and blood lactate (LA) concentrations were determined in venous blood samples as well as plasma growth hormone (HGH) and cortisol concentrations before and at the end of exercise. A decrease in NA concentration was found already after 1 week of training at power output of 100 W (p<0.01) and 200 W (p<0.05). Similar decline was maintained after 3 weeks of training. No significant training-induced differences in plasma A concentration were found, however, the thresholds for both catecholamines were significantly shifted towards higher values after 3 weeks of training. One week of training caused a decrease in the pre-exercise (p<0.01), as well as post-exercise (p<0.05) plasma cortisol and HGH concentrations. It was concluded that endurance training induced a decrease in HGH, cortisol and NA concentration already after one week of training. A decline of pre-exercise plasma HGH and cortisol levels with time of experiment may, in part, indicate familiarization to exercise protocol.

Exercise training and 3-day head down bed rest deconditioning: exercise thermoregulation.

UNLABELLED: Bed rest (BR) deconditioning causes excessive increase of exercise core body tempera-ture, while aerobic training improves exercise thermoregulation. The study was designed to determine whether 3 days of 6 degrees head-down bed rest (HDBR) affects body temperature and sweating dynamics during exercise and, if so, whether endurance training before HDBR modifies these responses. Twelve healthy men (20.7+/-0.9 yrs, VO2max: 46+/-4 ml x kg(-1) x min(-1) ) underwent HDBR twice: before and after 6 weeks of endurance training. Before and after HDBR, the subjects performed 45 min sitting cycle exercise at the same workload equal to 60% of VO2max determined before training. During exercise the VO2, HR, tympanic (Ttymp) and skin (Tsk) temperatures were recorded; sweating dynamics was assayed from a ventilated capsule on chest. Training increased VO2max by 12.1% (p<0.001). Resting Ttymp increased only after first HDBR (by 0.22 +/- 0.08 degrees C, p<0.05), while exercise equilibrium levels of Ttymp were increased (p<0.05) by 0.21 +/- 0.07 and 0.26 +/- 0.08 degrees C after first and second HDBR, respectively. Exercise mean Tsk tended to be lower after both HDBR periods. Total sweat loss and time-course of sweating responses were similar in all exercise tests. The sweating threshold related to Ttymp was elevated (p<0.05) only after first HDBR. IN CONCLUSION: six-week training regimen prevents HDBR-induced elevation of core temperature (Ttymp) at rest but not during ex-ercise. The post-HDBR increases of Ttymp without changes in sweating rate and the tendency for lower Tsk suggest an early (<3d) influence of BR on skin blood flow.

Early effects of short-term aerobic training. Physiological responses to graded exercise.

AIM: The aim of this study was to find out how early the moderate training effects appear and to check the hypothesis that familiarization with exercise protocol may contribute to an early physiological responses to training in previously sedentary subjects. METHODS: Twelve male, sedentary volunteers (22.0+/-0.7 yrs) were submitted to 3 weeks of a bicycle ergometer training, consisting of 45 min of exercise (at 70% VO(2)max), 3-4 times a week. The subjects performed 4 incremental exercise tests until volitional exhaustion: 2 before training (C1 and C2), and then after 1 (T1) and 3 (T3) weeks of training. During exercise HR, VO(2), electrical activity (EMG) of rectus femoris, biceps femoris, soleus and trapezius muscles were recorded and blood samples were taken for blood lactate (LA) determination. RESULTS: Already after 1 week of training HR decreased (p<0.05) with a further decline after 3 weeks the training (p<0.01). Maximal work load after 3 weeks of training increased to 277+/-10.4 W vs 250+/-9.5 W (p<0.05), VO(2)max achieved higher values than in C1 and C2 tests (p<0.05) and LA and EMG thresholds were elevated (p<0.05). CONCLUSION: A decrease in the resting and submaximal heart rate is the earliest effect of increased physical activity. Familiarization to exercise protocol decreased EMG of biceps femoris and soleus muscles during exercise, but did not influence that of rectus femoris muscle the most engaged during cycling.

Repair of triplex-directed DNA alkylation by nucleotide excision repair.

Triplex-forming oligonucleotides (TFOs) are being investigated as highly specific DNA binding agents to inhibit the expression of clinically relevant genes. So far, they have been shown to inhibit transcription from the HER-2/neu gene in vitro, whereas their use in vivo has been studied to a limited extent. This study uses a TFO-chlorambucil (chl) conjugate capable of forming site-specific covalent guanine adducts within the HER-2/neu promoter. We demonstrate that nucleotide excision repair (NER) represents a mechanism of cellular resistance to TFO-directed DNA alkylation. In vitro repair assays demonstrate that triplex-directed chl-guanine adducts are substrates for repair by NER competent cell extracts but not XP12BE cell extracts deficient in NER. The degree of repair is estimated by a ligation-mediated polymerase chain reaction with a pre-formed triplex in a plasmid transfected into repair competent cells, indicating that approximately 25% of the guanine adducts are removed after 24 h. These data indicate that guanine adducts from TFO-directed alkylation are a substrate for NER and that DNA repair is a significant barrier to the intracellular persistence of target gene binding by TFOs.

Influence of caffeine, cold and exercise on multiple choice reaction time.

RATIONALE: The effects of caffeine on psychomotor performance have been evaluated under resting conditions and in a thermoneutral environment. Our hypothesis was that these effects could be modified by factors enhancing the level of alertness, such as exercise and cold exposure. OBJECTIVE: The purpose of this study was to follow up changes in the multiple choice reaction time (RT) during exercise at room and low ambient temperatures after caffeine or placebo administered in a double blind manner. METHODS: Nine soccer players performed multistage, incremental exercise until volitional exhaustion on a bicycle ergometer at 22 degrees C or 4 degrees C, 1 h after ingestion of coffee with caffeine (CAF) or without it (PL). Immediately before exercise and at the end of each workload, RT and blood lactate (LA) were measured. Oxygen uptake (VO2) and heart rate (HR) were recorded continuously. Blood LA threshold and the workload associated with the shortest RT were determined. RESULTS: During exercise at 22 degrees C, RT was significantly shorter in CAF than in the PL test, while at 4 degrees C there were no differences in RT between CAF and PL trials. Cold exposure did not affect RT either at rest or during exercise. Neither caffeine nor cold exposure influenced the maximal VO2, the maximal HR and LA threshold. CONCLUSION: In the thermoneutral environment, caffeine ingestion improved psychomotor performance during exercise, whilst at low ambient temperature this effect was blunted. These findings suggest that the stimulating action of caffeine depends on the level and source of arousal.

Cardiovascular, metabolic and plasma catecholamine responses to passive and active exercises.

Eight healthy male volunteers (aged 19.6+/-3.0 years) were submitted to the unloaded active (AE) and passive (PE) cycling exercise-tests performed on an adapted cycle ergometer at a pedalling rate of 50 rpm. Intensity of active exercise was about 10% of VO2 max. In the PE exercise test the ergometer was moved electrically. During both tests the systolic time intervals (STI), stroke volume (SV), heart rate (HR), blood pressure (BP), oxygen uptake (VO2), rating of perceived exertion (RPE), electrical muscle activity (EMG), plasma adrenaline (A), noradrenaline (NE) and blood lactate (LA) concentrations were measured. Exercise induced changes in VO2, RPE and EMG were significantly higher during AE than PE. Shortening of the pre-ejection period (PEP) and diminishing of the PEP to ejection time (ET) ratio were similar in both types of exercise, whereas HR increased only during AE. A significant increase in cardiac output (p<0.01) resulted from increased SV (p<0.01) during PE and from increased HR (p <0.01) during AE. MAP increased only during PE and it was higher than at rest and during AE (p<0.01). Absence of changes in SV and MAP during AE may be considered as a secondary effect of the decrease in TPR. Plasma catecholamines did not increase above resting values in either type of exercise. Blood LA concentration increased during both PE and AE but it reached higher values (p<0.01) after the latter test. The present data suggest that the inotropic state depends on the mechanoreflexes originated in skeletal muscles. However, contribution of changes in preload to shortening of PEP can not be excluded.

Effects of three-day bed rest on metabolic, hormonal and circulatory responses to an oral glucose load in endurance or strength trained athletes and untrained subjects.

The study was designed to find out (1) whether the effect of 3-day bed rest on blood glucose (BG) and plasma insulin (IRI) responses to glucose ingestion depends on preceding physical activity and (2) whether plasma adrenaline (A), noradrenaline (NA) and cardiovascular changes following a glucose load are modified by bed rest. Eleven sedentary students (22.5+/-0.3 yrs), 8 long distance runners (18.6+/-0.3 yrs) and 10 strength trained athletes (21.2+/-2.1 yrs) were examined before and after bed rest. Plasma IRI, BG, NA, A, heart rate (HR), and blood pressure (BP) were measured during 2 hrs following glucose (75 g) ingestion. The responses of BG and IRI to glucose load were calculated as incremental areas under the curves (auc). Both in athletes and untrained subjects bed rest markedly increased IRIauc, while BGauc was elevated only in sedentary subjects (p<0.05). The greatest increases in IRIauc and IRI/BG ratios were found in the endurance athletes. The data from all subjects (n = 29) revealed that the initial plasma NA and glucose-induced increases in NA and A were lowered after bed rest (p < 0.01). These effects were most pronounced in the endurance athletes. Bed rest did not influence HR or BP in any group. It is concluded that (1) the athletes have more adequate compensation for the bed-rest-induced decrement in insulin sensitivity than sedentary men; (2) three-day bed rest diminishes basal sympathetic activity and attenuates sympathoadrenal response to oral glucose; (3) endurance athletes have greater sympathetic inhibition than strength athletes or sedentary men.

Nuclear DEAF-1-related (NUDR) protein contains a novel DNA binding domain and represses transcription of the heterogeneous nuclear ribonucleoprotein A2/B1 promoter.

Nuclear DEAF-1-related (NUDR) protein is a novel transcriptional regulator with sequence similarity to developmental and oncogenic proteins. NUDR protein deletions were used to localize the DNA binding domain between amino acids 167 and 368, and site-specific DNA photocross-linking indicated at least two sites of protein-DNA contact within this domain. The DNA binding domain contains a proline-rich region and a region with similarity to a Myc-type helix-loop-helix domain but does not include the zinc finger motif at the C terminus. Deoxyribonuclease I protection assays confirmed the presence of multiple NUDR binding motifs (TTC(C/G)G) in the heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) promoter and also in the 5'-untranslated region (UTR) of hNUDR cDNA. NUDR produced a 65-70% repression of the hnRNP A2/B1 promoter activity, and NUDR binding motifs in the 5'-UTR were found to mediate this repression. NUDR-dependent repression was also observed when the 5'-UTR of NUDR was placed onto a heterologous thymidine kinase promoter in an analogous 5'-UTR position but not when placed upstream of transcription initiation. These results suggest that NUDR may regulate the in vivo expression of hnRNP A2/B1 and NUDR genes and imply that inactivation of NUDR could contribute to the overexpression of hnRNP A2/B1 observed in some human cancers.

Ginseng treatment improves psychomotor performance at rest and during graded exercise in young athletes.

This study was designed to determine the effect of ginseng treatment on multiple choice reaction time (RT) during exercise. Fifteen soccer players (age 19.07 +/- 0.62 yrs) were placed in a double-blind manner into one of two groups: ginseng (n = 7), receiving 350 mg of ginseng daily for 6 weeks, or placebo (n = 8), receiving a placebo daily for 6 weeks. Before and after the treatment all the subjects performed an incremental bicycle ergometer exercise with intensity increasing 50 W every 3 min until volitional exhaustion. RT was measured before exercise, and then in the last 2 min of each exercise load. Maximal oxygen uptake (VO2max) and lactate threshold (LAT) were also determined from the exercise test. Ginseng treatment was found to shorten RT at rest and during exercise, shifting the exercise load associated with the shortest RT toward higher exercise loads. Neither ginseng nor placebo influenced VO2 max and LAT. In conclusion, ginseng extract does improve psychomotor performance during exercise without affecting exercise capacity.

Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein.

A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Relationship between EMG blood lactate, and plasma catecholamine thresholds during graded exercise in men.

The aim of this study was to follow up the electromyographic activity (EMG) of dynamically working muscles with simultaneous determinations of blood lactate and plasma catecholamine concentrations during progressive exercise. Twenty eight male soccer players aged 20.6 +/- 0.8 yrs performed incremental bicycle ergometer exercise test. The test consisted of 3-min stages exercise separated by 1-min rest intervals. Work load at each stage increased by 50 W until volitional exhaustion. The root mean square (rms)-EMG activity of the rectus femoris and soleus muscles were recorded continuously during exercise. Venous blood samples were taken after each exercise stage for determination of blood lactate (LA). Additionally in seven subjects adrenaline (A) and noradrenaline (NA) concentrations were determined. The EMG activity increased negligibly during exercise of low to moderate intensities revealing an abrupt rise at the load corresponding to thresholds of blood lactate and plasma catecholamine accumulation (LA-T, A-T, NA-T). Close correlations (P < 0.001) were found between blood LA concentration and EMG derived from rectus f. (r = 0.72) and soleus (r = 0.68) muscles. The mean threshold exercise intensities for m. rectus f. and m. soleus EMG (176 +/- 9 W and 172 +/- 9 W, respectively) did not differ significantly from lactate (164 +/- 7 W), noradrenaline (178 +/- 6 W) and adrenaline (180 +/- 5 W) thresholds, all of them detected by log-log transformation. The results indicate that threshold character of EMG changes in dynamically working muscles reflects to some extend the patterns of blood lactate and plasma catecholamine changes during incremental exercise.

Psychomotor performance during prolonged exercise above and below the blood lactate threshold.

Previous investigations from this laboratory have demonstrated that during graded exercise with exercise intensities increasing every 3 min until exhaustion the multiple choice reaction time (RT) decreased until the intensity exceeded the lactate threshold (LT) by approximately 25%, and then rapidly increased. The aim of this study was to follow up changes in RT during prolonged exercise at constant intensities above and below LT and to relate these changes to changes in venous blood lactate [La-]b, and plasma catecholamine [CA]pl concentration responses to the exercise. For this purpose eight young soccer players exercised for 20-min on a cycle ergometer at 10% above LT, and nine exercised for 60 min at an intensity 30% below LT. During both tests RT, heart rate (HR), as well as [La-]b, and [CA]pl were measured. Above LT, RT decreased from the 5th min until the end of exercise, whilst HR, [La-]b, and [CA]pl increased progressively. Significant inverse correlations were ascertained between RT and plasma adrenaline (r = -0.651) and noradrenaline concentrations (r = -0.678). During exercise below LT, RT decreased up to approximately 40 min, then it reached a nadir, and stabilized at this level. This was accompanied by only small changes in [La-]b and [CA]pl. The present findings would indicate that young athletes are able to maintain for a relatively long time, or even increase, their psychomotor performance during endurance exercise both below and above the LT.