Effect of administration of oral contraceptives on the synthesis and breakdown of myofibrillar proteins in young women.

Oral contraceptive (OC) treatment has an inhibiting effect on protein synthesis in tendon and muscle connective tissue. We aimed to investigate whether OC influence myofibrillar protein turnover in young women. OC-users (24±2 years; Lindynette® n=7, Cilest® n=4) and non-OC-users (controls, 24±4 years n=12) performed one-legged kicking exercise. The next day, the myofibrillar protein fractional synthesis rate (FSR) was measured using stable isotopic tracers ((13)C-proline) while the subjects were fed standardized nutrient drinks. Simultaneously, a marker for myofibrillar protein breakdown, 3-methyl-histidine (3-MH), was measured in the interstitial fluid of the vastus lateralis. Measurements were performed in both legs. In general, myofibrillar protein FSR was lower in OC-users (two-way analysis of variance, P<0.05), although the difference seemed to depend on the OC type. Interstitial 3-MH in the skeletal muscle was not different between groups and did not vary by OC type. Exercise did not change myofibrillar protein FSR or 3-MH concentrations. Serum androstenedione and bioavailability of testosterone were lower in OC-users. In conclusion, the results indicate that the use of OC has an inhibiting effect on myofibrillar protein synthesis and the magnitude of the effect may depend on the type of OC. In contrast, there was no effect of OC on myofibrillar protein breakdown in the fed state.

Measurement of skeletal muscle collagen breakdown by microdialysis.

Exercise increases the synthesis of collagen in the extracellular matrix of skeletal muscle. Breakdown of skeletal muscle collagen has not yet been determined because of technical limitations. The purpose of the present study was to use local sampling to determine skeletal muscle collagen breakdown. Microdialysis fibers were tested in vitro to predict bath hydroxyproline (OHP) concentrations. We used an N-methyl-N-[tert-butyldimethyl-silyl]trifluoroacetimide derivative to analyze OHP using gas chromatography-mass spectroscopy (GC-MS) and compared the results with a colorimetric OHP assay. Ten young, healthy male subjects performed a bout of resistance exercise with one leg, followed 17-21 h later by in vivo skeletal muscle sampling by microdialysis in exercised (EX) and control (CON) legs. Microdialysis reliably predicted [OHP] in vitro (R(2)=0.90). Analysis with GC-MS was strongly correlated to traditional analysis methods (CON: slope=1.03, R(2)=0.896, and P<0.05, EX: slope=0.795, R(2)=0.896, and P<0.05). We conclude that in vitro, microdialysis fibers were able to measure OHP concentrations and were sensitive to changes in concentrations, a strenuous bout of exercise did not increase skeletal muscle collagen breakdown 17-21 h post-exercise, and our measurement of OHP using GC-MS was in agreement with traditional assays.

Energy intake and expenditure during a 6-day cycling stage race.

Energy intake (EI) and energy expenditure (EE) are relatively easy to measure accurately over short periods in a laboratory setting, but less so during a multi-day competition. Our goal was to measure EI and EE as accurately as possible during a 6-day, 10-stage cycling race. We prepared all meals and supplements, assessed EI (weighed diet-records) and macrontrient intake, total EE (doubly labelled water), resting metabolic rate (respiratory gas exchange), exercise EE (power meters), and body mass. Body composition was measured several days before and after racing (dual x-ray absorptiometry). Body mass remained stable over the course of the race. The mean EI (27.3+/-3.8 MJ/day) nearly matched EE (27.4+/-2.0 MJ/day). The majority (62%) of EE was exercise EE. Macronutrient intake was within or exceeded the recommendations. Lean body mass increased and fat mass decreased in most of our participants. Our study indicates that EI can match high EE with adequate macronutrient intake during multi-day cycle racing and may be facilitated by appropriate foods being available at appropriate times. This optimization of nutritional provision supports positive changes in body composition.

Effect of administration of oral contraceptives in vivo on collagen synthesis in tendon and muscle connective tissue in young women.

Women are at greater risk than men for certain kinds of diseases and injuries, which may at least partly be caused by sex hormonal differences. We aimed to test the influence of estradiol in vivo on collagen synthesis in tendon, bone, and muscle. Two groups of young, healthy women similar in age, body composition, and exercise-training status were included. The two groups were either habitual users of oral contraceptives exposed to a high concentration of synthetic estradiol and progestogens (OC, n = 11), or non-OC-users tested in the follicular phase of the menstrual cycle characterized by low concentrations of estradiol and progesterone (control, n = 12). Subjects performed 1 h of one-legged kicking exercise. The next day collagen fractional synthesis rates (FSR) in tendon and muscle connective tissue were measured after a flooding dose of [(13)C]proline followed by biopsies from the patellar tendon and vastus lateralis in both legs. Simultaneously, microdialysis catheters were inserted in vastus lateralis and in front of the patellar tendon for measurement of insulin-like growth factor I (IGF-I) and its binding proteins. Serum NH(2)-terminal propeptide of type I collagen (PINP) and urine COOH-terminal telopeptides of type-I collagen (CTX-I) were measured as markers for bone synthesis and breakdown, respectively. Tendon FSR and PINP were lower in OC compared with control. An increase in muscle collagen FSR postexercise was only observed in control (P < 0.05). Furthermore, the results indicate a lower bioavailability of IGF-I in OC. In conclusion, synthetic female sex hormones administered as OC had an inhibiting effect on collagen synthesis in tendon, bone, and muscle connective tissue, which may be related to a lower bioavailability of IGF-I.

The effect of strenuous aerobic exercise on skeletal muscle myofibrillar proteolysis in humans.

Relatively little is known about the dynamics of the skeletal muscle protein pool following aerobic exercise. Myofibrillar protein synthesis has recently been shown to be substantially elevated for 3 days after a strenuous 60 min bout of one-legged aerobic exercise, and this increase was surprisingly equal to or greater than what has been shown numerous times following resistance exercise over the same time course. Because net protein accretion is the sum of protein synthesis and degradation, we sought to directly measure skeletal muscle myofibrillar proteolysis in five healthy young males in response to an identical strenuous 60 min aerobic exercise bout and at the same time points (rest, 6, and 24 h post-exercise and 48 and 72 h post-exercise in a subset of subjects). We measured skeletal muscle myofibrillar proteolysis by monitoring the release of the natural tracer 3-methylhistidine (3MH) from the vastus lateralis muscle into the interstitial space via microdialysis. Skeletal muscle interstitial 3MH concentration was no different (P>0.05) from rest (5.16+/-0.38 nmol/mL) after 6 (5.37+/-0.55 nmol/mL), 24 (5.40+/-0.26 nmol/mL), 48 (5.50+/-0.74 nmol/mL), or 72 h (4.73+/-0.28 nmol/mL). These results suggest that proteolysis of the myofibrillar fraction of skeletal muscle is relatively refractory to an intense aerobic exercise stimulus for up to 3 days, despite the large increase in synthesis of this muscle fraction following the same exercise stimulus. The apparent net myofibrillar protein accretion in the hours and days after exercise may occur in order to offset the large elevation in mixed muscle proteolysis that has been shown during similar bouts of intense one-legged aerobic exercise.

Metabolic activity and collagen turnover in human tendon in response to physical activity.

Connective tissue of the human tendon plays an important role in force transmission. The extracellular matrix turnover of tendon is influenced by physical activity. Blood flow, oxygen demand, and the level of collagen synthesis and matrix metalloproteinases increase with mechanical loading. Gene transcription and especially post-translational modifications of proteins of the extracellular matrix are enhanced following exercise. Conversely, inactivity markedly decreases collagen turnover. Training leads to a chronically increased collagen turnover, and dependent on the type of collagen also to some degree of net collagen synthesis. These changes modify the biomechanical properties of the tissue (for example, viscoelastic characteristics) as well as the structural properties of the in collagen (for example, cross-sectional area). Mechanical loading of human tendon does result in a marked interstitial increase in growth factors that are known potentially to stimulate synthesis of collagen and other extracellular matrix proteins. Taken together, human tendon tissue mounts a vigorous acute and chronic response to mechanical loading in terms of metabolic-circulatory changes as well as of extracellular matrix formation. These changes may contribute to training-induced adaptation of biomechanical properties consisting of altered resistance to loading and enhanced tolerance to strenuous exercise. Understanding of such changes is a pre-requisite in the development of measures aimed at prevention of overuse tendon injuries occurring during sport, work or leisure-related activities.

Circulatory responses to voluntary and electrically induced muscle contractions in humans.

BACKGROUND AND PURPOSE: Transcutaneous electrical nerve stimulation (TENS) increases regional blood flow when applied at intensities sufficient to cause skeletal muscle contraction. It is not known whether increases in blood flow elicited by TENS differ from those caused by voluntary muscle contraction. The purpose of this study, therefore, was to compare the hemodynamic effects of these 2 types of muscle contraction. SUBJECTS AND METHODS: Fourteen people with no known pathology, aged 18 to 49 years (mean=28, SD=8), served as subjects. Calf blood flow (venous occlusion plethysmography), heart rate (electrocardiogram), blood pressure (automated sphygmomanometry), and force (footplate transducer) were measured during electrically induced and voluntary contractions. RESULTS: Both modes of exercise caused rapid, but short-lived vasodilation (calf vascular resistance [mean(SEM]: (53%(3% for voluntary contractions versus (57%(4% for electrically induced contractions). The vasodilation caused by electrically induced contractions persisted for at least 15 seconds in the postexercise period, whereas the vasodilation elicited by voluntary contractions had resolved by this time point. CONCLUSION AND DISCUSSION: The hemodynamic changes elicited by voluntary and electrically induced muscle contractions are similar in magnitude but different in duration.

Mucosal unresponsiveness to aflatoxin B1 is not broken by cholera toxin.

Rabbits immunized via chronically isolated ileal loops with aflatoxin B1 (AFB) conjugated to porcine thyroglobulin (TG) mixed with the mucosal adjuvant cholera toxin (CT) produced very small mucosal antibody responses to AFB. Strong mucosal and systemic antibody responses to CT and TG were generated by this immunization protocol, suggesting that the observed unresponsiveness was specific to AFB. Parenteral immunization with AFB-TG produced strong serum IgG anti-AFB responses, indicating that the conjugate preparation was immunogenic and that the rabbits possess the requisite systemic B and T cell repertoires to recognize and respond to AFB. This mucosal unresponsiveness was distinct from oral tolerance, as animals immunized mucosally with AFB-TG mixed with CT produced vigorous serum IgG anti-AFB responses upon subsequent parenteral immunization with AFB-TG. In vitro mitogen stimulation of lymphocytes isolated from Peyer's patches and mesenteric lymph nodes of unimmunized rabbits revealed the presence of AFB-specific B cells at levels comparable with these found in the spleen. These observations indicate that unresponsiveness to AFB is hapten-specific, restricted to the mucosa, and refractory to the adjuvancy of CI.

The membrane attack complex of complement induces interleukin-8 and monocyte chemoattractant protein-1 secretion from human umbilical vein endothelial cells.

Cell surface assembly of the membrane attack complex (MAC) of complement occurs in a variety of pathophysiological settings. Depending upon the density and size distribution of pores formed by the MAC and the functional integrity of membrane regulators of complement activation, the MAC can either cause direct cell lysis or transduce cell activation. We have examined the functional capacity of sublytic concentrations of MAC to induce the secretion of specific alpha- and beta-chemokines from human umbilical vein endothelial cells (HUVECs). Endothelial cell activation by the MAC has particular relevance to complement-dependent inflammatory processes including ischemia-reperfusion injury and acute lung injury. Assembly of sublytic concentrations of the MAC on HUVECs resulted in the sequential secretion of both neutrophil and monocyte chemotactic activities. Analysis of conditioned medium from MAC-bearing HUVECs revealed that the neutrophil chemotactic activity was largely attributable to interleukin (IL)-8, whereas the monocyte chemotactic activity, which was detected later (peak at 8 hours versus 4 hours), was largely attributable to MCP-1. This temporal pattern of MAC-induced secretion of IL-8 and MCP-1 was confirmed using IL-8- and MCP-1-specific enzyme-linked immunosorbent assays. Northern hybridization analysis of HUVECs revealed that MAC deposition was accompanied by an increase in IL-8 and MCP-1 mRNA levels. These data indicate that assembly of sublytic concentrations of the MAC on HUVECs can induce the sequential secretion of both neutrophil and monocyte chemotactic activities and that the former is largely attributable to IL-8 whereas the latter is largely attributable to MCP-1.

Antibodies to neutrophil cytoplasmic antigens induce monocyte chemoattractant protein-1 secretion from human monocytes.

Antibodies to neutrophil cytoplasmic antigens (ANCA) have been found in the serum samples of patients with a number of vasculitides (e.g., Wegener's granulomatosis, small vessel vasculitis, and idiopathic necrotizing and cresentic glomerulonephritis). Although detection of ANCA in serum samples has proven to be useful diagnostically and in selected activity of disease monitoring situations, the pathogenetic role of ANCA in vasculitis remains ill-defined. We sought to determine whether purified ANCA promotes the secretion of monocyte chemoattractant protein-1 (MCP-1) from isolated human peripheral blood monocytes. P (perinuclear)- and C (cytoplasmic)- ANCA were purified from the serum samples of patients with either Wegener's granulomatosis, small vessel vasculitis, or idiopathic necrotizing and cresentic glomerulonephritis. Human peripheral blood monocytes from healthy subjects were incubated with either C-ANCA immunoglobulin G (IgG), P-ANCA IgG, or nonspecific IgG, and the conditioned media were analyzed for MCP-1 activity. A monocyte chemotaxis assay was utilized to functionally quantify secreted chemotactic activity. Secretion of monocyte chemotactic activity was found to be antibody concentration-dependent and time-dependent, with maximal chemotaxis measured in media collected 24 hours after the addition of either C- or P-ANCA IgG. A specific antibody directed against human MCP-1 largely inhibited monocyte chemotaxis, indicating that MCP-1 is the predominant monocyte chemotactic mediator present in the conditioned medium. An MCP-1 enzyme-linked immunosorbent assay further supported the conclusion that P- and C-ANCA IgG can trigger MCP-1 secretion by monocytes. These data indicate that incubation of monocytes with ANCA promotes the dose-dependent release of the chemotactic beta-chemokine MCP-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhancement by the complement membrane attack complex of tumor necrosis factor-alpha-induced endothelial cell expression of E-selectin and ICAM-1.

Although TNF-alpha and several products of the activated complement system (e.g., C3b, iC3b, and C5a) are known to modulate endothelial cell function in vitro, relatively little is known about the potential modulatory role of the membrane attack complex (MAC) in endothelial cell activation. Using an in vitro neutrophil-endothelial adhesion assay and a quantitative whole cell ELISA to measure endothelial E-selectin and intracellular adhesion molecule-1 (ICAM-1) expression, we examined the modulatory role of the MAC in TNF-alpha-induced neutrophil-endothelial cell adhesive interactions. Activation of quiescent human umbilical vein endothelial cells (HUVECs) with TNF-alpha results in a concentration-dependent increase in neutrophil adhesion measured at 4 h. Assembly of sublytic concentrations of the MAC on endothelial cells did not result in changes in neutrophil-HUVEC adhesion measured at 4 h. Activation of HUVECs with TNF-alpha followed by assembly of the MAC resulted in a marked increase in neutrophil binding as compared with that observed in cells treated with TNF-alpha alone. Blocking studies of mAb revealed that in either TNF-alpha-stimulated or TNF-alpha and MAC-activated endothelial cells enhanced neutrophil binding was nearly entirely attributable to E-selectin and ICAM-1. This conclusion was further supported by a whole-cell ELISA, which provided evidence that the MAC augments TNF-alpha-induced up-regulation of both E-selectin and ICAM-1. This study provides data that support the conclusion that the distal complement system (MAC) can enhance TNF-alpha-induced proinflammatory endothelial cell functions.

Regulatory roles of tumor necrosis factor-alpha and interleukin-1 beta in monocyte chemoattractant protein-1-mediated pulmonary granuloma formation in the rat.

Intravenous infusion of particulate yeast cell wall glucan into rats results in the synchronous development of angiocentric pulmonary granulomas that are composed almost entirely of monocytes and macrophages. Previous studies indicate that locally produced monocyte chemoattractant protein-1 (MCP-1) is required for full granuloma development. Because tumor necrosis factor-alpha (TNF-alpha) and interleukin 1 (IL-1) can induce MCP-1 production in a variety of cell types, we sought to determine their potential regulatory roles in this model. A single infusion of anti-TNF-alpha antibody at the time of glucan infusion (time 0) markedly reduced MCP-1 mRNA levels at 1 and 6 hours but not at later time points; there was no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-TNF-alpha antibody were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a significant reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. Similar results were observed in animals that received infusions of anti-IL-1 beta. Infusion of anti-IL-1 beta at time 0 resulted in moderate reductions in MCP-1 mRNA at 1 and 6 hours and had no effect on granuloma size or number measured at 48 hours. When multiple infusions of anti-IL-1 beta were administered over a 23-hour period (0 to 23 hours), MCP-1 mRNA was reduced through 24 hours, there was a moderate reduction in peak bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and there were marked reductions in granuloma size and number at 48 hours. A single infusion of anti-TNF-alpha and anti-IL-1 beta together at time 0 resulted in marked reductions in whole lung MCP-1 and mRNA at 1 and 6 hours, but not at 24 hours. Multiple combined infusions of anti-TNF-alpha and anti-IL-1 beta over a 23-hour period resulted in additive reductions in MCP-1 mRNA through 24 hours, bronchoalveolar lavage fluid MCP-1 activity at 48 hours, and granuloma size and number at 48 hours. These data suggest that locally produced TNF-alpha and IL-1 beta play regulatory roles in glucan-induced pulmonary granulomatous vasculitis through the modulation of local MCP-1 production.

Use of UV ParaLens adapter for detection of acid-fast organisms.

Auramine-stained mycobacterial smears from 136 clinical specimens were interpreted by using the UV ParaLens adapter (Beckton Dickinson), and results were compared with smear interpretations using a traditional fluorescent microscope and culture. The sensitivity and specificity of the ParaLens were 84 and 93%, respectively. Smears yielding discrepant results were overstained by the Kinyoun method. Overall, the sensitivity of auramine-stained smears interpreted with the UV ParaLens was comparable to that of Kinyoun-stained smears.

Variation among inbred and linecross mice in response to fescue toxicosis.

Variation in response to fescue toxicosis was examined in inbred and linecross mice. In Exp. 1, exposure to a 50% endophyte-infected tall fescue diet (E+) reduced ADG of males from six inbred lines, but ADG of males from one line was modestly higher on E+. Lines differed (P < .01) for reproductive organ weight, but the diet x line interaction was not significant. In Exp. 2, an apparently susceptible (C57) and an apparently resistant line (FVB) were mated to produce inbred and linecross offspring. The reduction in weight gain caused by the E+ diet did not differ significantly among the genetic groups. In Exp. 3, C57 and C57 backcrosses had smaller reductions in ADG during E+ vs control feeding periods than FVB and FVB backcrosses (P < .10). In Exp. 4, the E+ diet reduced litter size of mates of C57 males by one pup, whereas litter size of mates of FVB males was four pups larger (interaction P = .07). Neither diet, line, nor their interaction affected male reproductive organ weights or tissue proportions in testis cross-sections. In Exp. 5, the E+ diet did not affect weight gain of C57 or FVB males, but effects of the E+ diet on litter size of mates were similar to those in Exp. 4. Percentage of abnormal sperm was increased in C57 males on the E+ diet but decreased in FVB males (Exp. 5). Differences among inbred lines in susceptibility to fescue toxicosis may depend on severity of the challenge and life cycle stage when the challenge is imposed.

Differences in activity of minoxidil and cyclosporin A on hair growth in nude and normal mice. Comparisons of in vivo and in vitro studies.

Hair growth effects of minoxidil and cyclosporin A were assessed in a series of experiments using nude mice. Systematic monitoring of coat hair showed that untreated nude mice grow extremely sparse and transient hair in cycles. This monitoring was done by photographing each animal through at least one full growth cycle and rating peak growth on a 1 to 4 scale. Topical administration of minoxidil or minoxidil sulfate did not influence this cyclic hair growth. Orally administered minoxidil also had no effect but oral cyclosporin A increased peak hair growth. None of the treatments altered the length of the hair cycle. Direct drug effects on follicles were tested in vitro using organ cultured vibrissae from both nude and normal mice. Minoxidil stimulated hair growth in follicles from normal but not nude mice. In contrast, cyclosporin A stimulated growth only in vibrissae follicles from nude but not normal animals. These studies show that minoxidil and cyclosporin A influence hair growth differentially. Cyclosporin A directly affects nude hair follicles by apparently compensating for a genetic defect inherent in nude follicles. Minoxidil does not have a similar effect. Apparently, the biochemical pathway activated by minoxidil is not a critical defect of hair growth in nude mice. We conclude that nude mice are not useful for studying minoxidil effects but they may be useful in studying pleiotropic effects of the nude gene on hair growth.

Colonization of Lactobacillus acidophilus in gnotobiotic chicks.

Germ-free chicks hatched in flexible film gnotobiotic isolators were dosed orally at 2 days of age with a pure culture of Lactobacillus acidophilus. The chicks monoassociated with L. acidophilus were killed at 2 weeks of age to obtain gastrointestinal tract specimens for histological sectioning. Both light microscopy and transmission electron microscopy were performed to determine colonization and adhesion of lactobacilli. This strain of L. acidophilus was found associated with epithelium from three segments of the gastrointestinal tract (crop, proventriculus, and duodenum) of the gnotobiotic chick. The electron micrographs showed not only a close relationship between the Lactobacillus organism and the crop epithelia, but also an attachment through physical contact.

Competitive gut exclusion of avian pathogens by Lactobacillus acidophilus in gnotobiotic chicks.

A total of 205 Grey Leghorn chicks were hatched germfree for separate trials to determine the effects of Lactobacillus acidophilus treatment on pathogenic Salmonella typhimurium and Staphlococcus aureus. Prophylactic and therapeutic treatments with L. acidophilus were administered either before or after the pathogens were introduced. Prophylactic treatments significantly reduced chick mortality (P less than .01) and shedding of the pathogens (P less than .05). The L. acidophilus prophylactic treatments were also effective qualitatively in reducing the isolation of S. typhimurium and Staph. aureus from crop contents but not, to a great extent, from cecal or rectal contents of gnotobiotic chicks at postmortem. The average surface pH values for the crop, proventriculus, duodenum, cecum, and rectum for gnotobiotic chicks were 5.43, 5.02, 6.18, 6.56, and 6.71, respectively. The L. acidophilus treatments did not significantly affect surface pH of the various segments of the intestinal tract.

In vivo inhibitory effects of Lactobacillus acidophilus against pathogenic Escherichia coli in gnotobiotic chicks.

Chicks were hatched germfree in gnotobiotic isolators to determine the inhibitory effects of Lactobacillus acidophilus towards pathogeneic Escherichia coli in vivo. Twelve trials were conducted in two flexible film isolators utilizing a total of 221 chicks. One treatment consisted of inoculating 2-day-old chicks with L. acidophilus, then challenging with pathogenic E. coli with subsequent dosing with L. acidophilus. The other treatment consisted of challenging with the E. coli at 2 days of age, then subsequently dosing with L. acidophilus. Statistical analysis of the data showed initial dosing with L. acidophilus prevented excessive mortality when chicks were challenged with E. coli. Also, continued dosing with L. acidophilus lowered the pH in the crop, cecum, and rectum whether chicks were initially given L. acidophilus or E. coli. This strain of L. acidophilus was capable of competing with E. coli in the gut of gnotobiotic chicks.