CNTF genotype is associated with muscular strength and quality in humans across the adult age span.

The relationship between ciliary neurotrophic factor (CNTF) genotype and muscle strength was examined in 494 healthy men and women across the entire adult age span (20-90 yr). Concentric (Con) and eccentric (Ecc) peak torque were assessed using a Kin-Com isokinetic dynamometer for the knee extensors (KE) and knee flexors (KF) at slow (0.52 rad/s) and faster (3.14 rad/s) velocities. The results were covaried for age, gender, and body mass or fat-free mass (FFM). Individuals heterozygous for the CNTF null (A allele) mutation (G/A) exhibited significantly higher Con peak torque of the KE and KF at 3.14 rad/s than G/G homozygotes when age, gender, and body mass were covaried (P < 0.05). When the dominant leg FFM (estimated muscle mass) was used in place of body mass as a covariate, Con peak torque of the KE at 3.14 rad/s was also significantly greater in the G/A individuals (P < 0.05). In addition, muscle quality of the KE (peak torque at 3.14 rad x s(-1) x leg muscle mass(-1)) was significantly greater in the G/A heterozygotes (P < 0.05). Similar results were seen in a subanalysis of subjects 60 yr and older, as well as in Caucasian subjects. In contrast, A/A homozygotes demonstrated significantly lower Ecc peak torque at 0.52 rad/s for both KE and KF compared with G/G and G/A groups (P < 0.05). No significant relationships were observed at 0.52 rad/s between genotype and Con peak torque. These data indicate that individuals exhibiting the G/A genotype possess significantly greater muscular strength and muscle quality at relatively fast contraction speeds than do G/G individuals. Because of high positive correlations between fast-velocity peak torque and muscular power, these findings suggest that further investigations should address the relationship between CNTF genotype and muscular power.

Reductions in visceral fat during weight loss and walking are associated with improvements in VO(2 max).

The accumulation of visceral fat is independently associated with an increased risk for cardiovascular disease. The aim of this study was to determine whether the loss of visceral adipose tissue area (VAT; computed tomography) is related to improvements in maximal O(2) uptake (VO(2 max)) during a weight loss (250-350 kcal/day deficit) and walking (3 days/wk, 30-40 min) intervention. Forty obese [body fat 47 +/- 1 (SE) %], sedentary (VO(2 max) 19 +/- 1 ml. kg(-1). min(-1)) postmenopausal women (age 62 +/- 1 yr) participated in the study. The intervention resulted in significant declines in body weight (-8%), total fat mass (dual-energy X-ray absorptiometry; -17%), VAT (-17%), and subcutaneous adipose tissue area (-17%) with no change in lean body mass (all P < 0.001). Women with an average 10% increase in VO(2 max) reduced VAT by an average of 20%, whereas those who did not increase VO(2 max) decreased VAT by only 10%, despite comparable reductions in body fat, fat mass, and subcutaneous adipose tissue area. The decrease in VAT was independently related to the change in VO(2 max) (r(2) = 0.22; P < 0. 01) and fat mass (r(2) = 0.08; P = 0.05). These data indicate that greater improvements in VO(2 max) with weight loss and walking are associated with greater reductions in visceral adiposity in obese postmenopausal women.

Muscle quality. I. Age-associated differences between arm and leg muscle groups.

To determine the differences between arm and leg muscle quality (MQ) across the adult life span in men and women, concentric (Con) and eccentric (Ecc) peak torque (PT) were measured in 703 subjects (364 men and 339 women, age range 19-93 yr) and appendicular skeletal muscle mass (MM) was determined in the arm and leg in a subgroup of 502 of these subjects (224 men and 278 women). Regression analysis showed that MQ, defined as PT per unit of MM, was significantly higher in the arm ( approximately 30%) than in the leg across age in both genders (P < 0.01). Arm and leg MQ declined at a similar rate with age in men, whereas leg MQ declined approximately 20% more than arm MQ with increasing age in women (P

Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems.

Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

Rapid development of severe hyperplastic gastritis with gastric epithelial dedifferentiation in Helicobacter felis-infected IL-10(-/-) mice.

Interleukin (IL)-10 is a potent anti-inflammatory and immune-regulatory cytokine. Mice deficient in IL-10 production (IL-10(-/-)) develop a spontaneous inflammatory bowel disease, indicating that IL-10 is an important regulator of the mucosal immune response in vivo. To study the role of IL-10 in the host response to gastric Helicobacter infection, stomachs of IL-10(-/-) and wild-type mice were colonized with Helicobacter felis, as a model of human H. pylori infection. Within 4 weeks of H. felis infection, wild-type mice develop a mild, focal chronic gastritis. In contrast, H. felis-infected IL-10(-/-) mice develop a severe hyperplastic gastritis, characterized by a dense, predominantly mononuclear cell inflammation of the mucosa and submucosa and epithelial cell proliferation and dedifferentiation. Within 4 weeks of H. felis infection, there are striking alterations in the character of the gastric epithelium from IL-10(-/-) mice, including a profound loss of parietal and chief cells, focal de novo production of acidic mucins, and marked epithelial proliferation with disordered epithelial architecture. These findings indicate that, in the absence of IL-10, the inflammatory and immunological responses of the murine host to gastric colonization with Helicobacter is a rapidly evolving pathological process with features that mimic those associated with H. pylori infection in humans. H. felis-infected IL-10(-/-) mice may provide a model with which to investigate the cellular and molecular changes that stem from gastric infection with H. pylori.

Temporal specificity in adaptations to high-intensity exercise training.

OBJECTIVE: The purpose was to test the hypothesis that time to exhaustion and oxygen deficit in high-intensity exercise at a particular time of day would be influenced by training regularly at that time of day. METHODS: Over a 5-wk period, 12 college-age women performed 20 high-intensity exercise training sessions. On Mondays, they performed four 2-min bouts of cycling at 2.5 W x kg(-1) with 4-min recoveries; on Tuesdays and Thursdays, eight 1-min bouts at 3.0 W x kg(-1) with 2-min recoveries; and on Wednesdays, three 3-min bouts at 2.2 W x kg(-1) with 2-min recoveries. Six participants (a.m.-trained group) were randomly assigned to train in the morning (a.m.) and six others (p.m.-trained group) trained in the afternoon (p.m.). Upon completion of training, all participants were tested in both the a.m. and p.m. (random order) at the same times as training sessions had been scheduled. Tests involved exhaustive efforts at 2.6 W x kg(-1). RESULTS: Results of a repeated measures ANOVA revealed a significant time of day of training x time of day of testing interaction effect on time to exhaustion (F1,10=8.29, P=0.02). This suggested that the time of day of training affected the a.m.-p.m. pattern in time to exhaustion. Time to exhaustion for the a.m.-trained group was 398+/-258 s in the a.m. test and 351+/-216 s in the p.m. test (P=0.07). The p.m.-trained group had significantly higher values in the p.m. test compared with the a.m. test (422+/-252 s vs 373+/-222 s; P=0.03). There was also a significant interaction effect on oxygen deficit (F1,10=8.03, P=0.02). This suggested that the time of day of training affected the a.m.-p.m. pattern in anaerobic capacity. Oxygen deficit for the a.m.-trained group was 64+/-24 mL x kg(-1) in the a.m. test and 50+/-11 mL x kg(-1) in the p.m. test (P=0.10). The p.m.-trained group had significantly higher values in the p.m. tests (64+/-24 mL x kg(-1) vs 50+/-11 mL x kg(-1); P=0.01) compared to the a.m. tests. CONCLUSIONS: These results demonstrate that there is temporal specificity in training to increase work capacity in high-intensity exercise. Greater improvements can be expected to occur at the time of day at which high-intensity training is regularly performed.

Psychological responses to brief exhaustive cycling exercise in the morning and the evening.

The purpose of this investigation was to examine whether psychological responses to exhaustive exercise would vary if the exercise was performed in the AM or PM. Sixteen men completed the State-Trait Anxiety Inventory and the Profile of Mood States before and following two exhaustive exercise sessions in the AM (0630-0930h) and the PM (1700-2000h). Data were analyzed with a 2 (condition = incremental, constant) x 2 (time of day = AM, PM) x 2 (trial = pre, post) repeated measures ANOVA. Results indicated that there was a significant time of day effect (P<0.05), as well as a significant condition by trial interaction (P<0.05) for vigor. Vigor was found to he higher in the PM before the constant power exhaustive exercise session, and decrease significantly following the session. There was also a significant trial effect (P<0.05) for fatigue. Fatigue was found to increase following the constant power exhaustive exercise sessions in the AM and PM. There were no significant changes in state anxiety, tension, depression, anger confusion or the composite measure of mood following exhaustive exercise in the AM or the PM. It is concluded that anxiety and mood responses to brief exhaustive cycling exercise are similar whether the exercise is performed in the morning or evening.

Age and gender comparisons of muscle strength in 654 women and men aged 20-93 yr.

To assess age and gender differences in muscle strength, isometric, concentric (Con), and eccentric (Ecc) peak torque was measured in the knee extensors at a slow (0.52 rad/s) and fast (3.14 rad/s) velocity in 654 subjects (346 men and 308 women, aged 20-93 yr) from the Baltimore Longitudinal Study of Aging. Regression analysis revealed significant (P < 0.001) age-related reductions in Con and Ecc peak torque for men and women at both velocities, but no differences were observed between the gender groups or velocities. Age explained losses in Con better than Ecc peak torque, accounting for 30% (Con) vs. 19% (Ecc) of the variance in men and 28% (Con) vs. 11% (Ecc) in women. To assess age and gender differences in the ability to store and utilize elastic energy, the stretch-shortening cycle was determined in a subset of subjects (n = 47). The older women (mean age = 70 yr) showed a significantly greater enhancement in the stretch-shortening cycle, compared with men of similar age (P < 0.01) and compared with younger men and women (each P < 0.05). Both men and women showed significant declines in muscle quality for Con peak torque (P < 0.01), but no gender differences were observed. Only the men showed a significant decline in muscle quality (P < 0.001) for Ecc peak torque. Thus both men and women experience age-related losses in isometric, Con, and Ecc knee extensor peak torque; however, age accounted for less of the variance in Ecc peak torque in women, and women tend to better preserve muscle quality with age for Ecc peak torque. In addition, older women have an enhanced capacity to store and utilize elastic energy compared with similarly aged men as well as with younger women and men.

Substrate oxidation at rest and during exercise: effects of menstrual cycle phase and diet composition.

The estrogen hormones have been shown to be highly glycogenic as well as lipolytic in nature. It is unknown whether the metabolic actions of estrogens impact upon energy metabolism during exercise. The composition of prior diet, however, does affect exercise energy metabolism. This study examined the influence of menstrual cycle phase (mid-follicular [FP; low estrogen] vs. mid-luteal [LP; high estrogen]) and diet composition on the rate of substrate oxidation for carbohydrate (CHO) and lipid at rest and during various intensities of physical exercise. Nine subjects completed an experimental session under four different menstrual cycle-diet conditions: 1) FP following a 3-day high CHO diet [75% total caloric intake], 2) FP following a 3-day low CHO diet [35% total caloric intake], 3) LP following a 3-day high CHO diet, and 4) LP following a 3-day low CHO diet. In each of the experimental sessions substrate oxidation was determined at rest and during cycle ergometer exercise at intensities of 30, 50, and 70% VO2max, respectively. Statistically significant (p < 0.05) interaction effects on substrate oxidation due to the menstrual cycle phase and diet conditions were found at rest and during 30%-50% exercise. In general, CHO oxidation was lowest and lipid oxidation highest in the LP under a low CHO diet condition.

Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.

A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.