T-lymphocyte populations following a period of high volume training in female soccer players.

PURPOSE: To investigate the T-lymphocyte response to a period of increased training volume in trained females compared to habitual activity in female controls. METHODS: Thirteen trained female (19.8 ± 1.9 yrs) soccer players were monitored during a two-week long high volume training period (increased by 39%) and thirteen female untrained (20.5 ± 2.2 yrs) controls were monitored during two-weeks of habitual activity. Blood lymphocytes, collected at rest, were isolated before and after the two-week period. Isolated lymphocytes were assessed for the cell surface expression of the co-receptor CD28, a marker of T-lymphocyte naivety, and CD57 a marker used to identify highly-differentiated T-lymphocytes. Co-expression of these markers was identified on helper CD4(+) and cytotoxic CD8(+) T-lymphocytes. In addition a further population of γδ(+) T-lymphocytes were identified. Plasma was used to determine Cytomegalovirus (CMV) serostatus. RESULTS: No difference was observed in the T-lymphocyte populations following the two-week period of increased volume training. At baseline the number of total CD3(+), cytotoxic CD8(+), naïve (CD8(+) CD28(+) CD57(-)), intermediate (CD8(+) CD28(+) CD57(+)) T-lymphocytes and the number and proportion of γδ(+) T-lymphocytes were greater in the trained compared to the untrained females (p<0.05). The proportion of CD4(+)T-lymphocytes was greater in the untrained compared to the trained (p<0.05), in turn the CD4(+):CD8(+) ratio was also greater in the untrained females (p<0.05). Inclusion of percentage body fat as a covariate removed the main effect of training status in all T-lymphocyte sub-populations, with the exception of the γδ(+) T-lymphocyte population. 8% of the untrained group was defined as positive for CMV whereas 23% of the trained group was positive for CMV. However, CMV was not a significant covariate in the analysis of T-lymphocyte proportions. CONCLUSION: The period of high volume training had no effect on T-lymphocyte populations in trained females. However, baseline training status differences were evident between groups. This indicates that long-term exercise training, as opposed to short-term changes in exercise volume, appears to elicit discernible changes in the composition of the blood T-lymphocyte pool.

Better palpation of pain: reliability and validity of a new pressure pain protocol in TMD.

The purpose of this research was to employ a double-blind placebo control design of iontophoretically administered lidocaine to assess the test-retest reliability and validity of a magnitude matching procedure using a pressure algometer. In studies in the literature on assessment of pain in temporomandibular disorders (TMD), the pressure algometer has been found to be a reliable means of measuring pressure-pain threshold and a sensitive measure of treatment-response in the laboratory. However, previous research with this apparatus has not employed more sophisticated psychophysics. This study employs a magnitude matching psychophysical protocol to obtain a multidimensional pain report. Test-retest reliability over two occasions, 3-8 days apart, was found to be moderate for discriminability (r = .71, p < .01) but poor for response bias (r = .44). The validity study used iontophoresis as an anesthetic in a double-blind placebo and no-treatment control design. Although it was hypothesized that subjects in the anesthesia group would demonstrate reduced discriminability as compared with the control groups, no differences were found among the three groups on this measure. However, differences in response bias were found, with both the placebo control and no-treatment control groups differing from the experimental group but not from each other. Possible explanations and the implications of these findings are discussed.

Pain severity, negative affect, and microstressers as predictors of life interference in TMD patients.

The purpose of this study was to examine the relationships among negative affect (depression, anxiety, and anger), microstressors (hassles), temporomandibular disorder (TMD) pain severity, and life interference, using structural equations modeling. One hundred four subjects were recruited from the Parker Mahan Facial Pain Clinic at the University of Florida. Significant positive direct effects were found for paths between pain severity and life interference; pain severity and negative affect; and negative affect and life interference. These results are consistent with a learning or behavioral model of suffering and suggest that negative affect is an important mediating variable in the relationship between pain and life interference. Microstressors were not a significant predictor and may not be a relevant issue in the TMD population. The results suggest that the impact of chronic pain conditions is influenced by both pain and negative affect, and assessment and treatment of chronic TMD disorders may better benefit from a multidisciplinary approach.

Common reference coherence data are confounded by power and phase effects.

Coherence analysis of the EEG is used to study the coupling between cortical regions. High coherence between signals recorded at 2 electrodes is interpreted as evidence for neuroanatomic connections between the cortical areas underlying the electrodes. When common reference recordings are used, coherence measures the relationship between 2 time series, each of which is the difference between 2 signals measured at the scalp and is confounded by spectral power and phase at the recording and reference electrodes. Using multi-channel EEG data from 3 subjects, we illustrate the confounding of common reference data coherence computations and demonstrate the extreme effects of power and phase changes on coherence by simulating these changes in the EEG data. Common reference coherence data can be either inflated or deflated as a consequence of activity (i.e., spectral power) at the reference. Phase relationships among the reference and recording time series modulate the power effects on coherence. Both the power and phase effects can vary dramatically across frequencies, having profound and complicated effects on the shape of the coherence function. Based on these considerations, we conclude that common reference coherence data must be interpreted very cautiously and recommend that a new body of EEG coherence data must be gathered using reference-free recording methods before the utility of EEG coherence analysis for understanding brain function can be determined.

A fifteen-item modification of the Fuld Object-Memory Evaluation: preliminary data from healthy middle-aged adults.

A modification of the Fuld Object-Memory Evaluation (FOME) test was developed for use with middle-aged adults. To avoid the ceiling effect which occurs when the 10-item FOME is used with adults in this age group, we made the test more difficult by increasing the number of items to 15. We obtained preliminary data on the 15-item version of the test by administering it to 32 healthy, well-educated adults aged 35 to 55. This version of the test was difficult enough so that even high functioning adults needed multiple trials to memorize all items. No sex differences, and no significant correlations with education or age for any aspect of test performance were found. We present mean scores for recall on each trial, as well as averaged scores for storage, retrieval, consistent retrieval, and recall failure across five trials.

Contralateral visual masking may be an artifact.

There are serious methodological problems in studies which report contralateral visual masking. Contralateral masking occurs when detection of a hemifield target stimulus is impaired by a pattern-masking stimulus presented to the opposite hemifield. We demonstrate that, in studies which used positive stimuli (i.e., black letters on a white field), contralateral masking may be an artifact. Although we observed contralateral masking when positive stimuli were presented, there was no evidence of masking with negative stimuli (i.e., white letters on a black field). A special masking stimulus with a positive mask contralateral to the target and a black hemifield ipsilateral to the target also failed to produce masking. Contralateral masking in this experiment was due to the flash of light in the field ipsilateral to the target; it was this ipsilateral stimulation, rather than contralateral interference, which impeded target recognition.

[NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella].

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T1 relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (delta H not equal to ) were determined for glycerol at 25 degree C: for phosphatidylcholine vesicles 5 . 10-6 cm/s and 11 +/- 2 kcal/mol; for pig erythrocytes 7 . 10-8 cm/s and 18 +/- 3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17 degrees C was found to be exceptionally low and only a lower limit (P less than 5 . 10-11 cm/s) could be calculated. At temperatures above 50 degree C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.

Plasma epinephrine and norepinephrine levels during anesthesia: enflurane-N2O-O2 compared with fentanyl-N2O-O2.

During intra-abdominal surgery, plasma levels of norepinephrine in peripheral venous blood were higher in 11 patients who received fentanyl-nitrous oxide-oxygen than in 10 patients who received enflurane-nitrous oxide-oxygen [703 +/- 95 vs 463 +/- 38 (SEM) pg/ml]. At the same time, systolic blood pressure (143 +/- 6 vs 121 +/- 4 torr), mean blood pressure (108 +/- 4 vs 98 +/- 3 torr), and pulse rate (87 +/- 3 vs 98 +/- 4 beats per minute) also differed significantly (p less than 0.05); plasma levels of epinephrine (235 +/- 61 vs 113 +/- 21) did not. These values did not differ significantly between the two groups before induction of anesthesia, after induction but before skin incision, or in the recovery room. These data support the concept that enflurane anesthesia blocks the sympathetic response to surgical stress more effectively than low dose fentanyl anesthesia.

Studies of lactate dehydrogenase in the purified state and in intact erythrocytes.

Lactate dehydrogenase in intact erythrocytes was studied by observing isotope exchange between lactate and pyruvate by p.m.r. The inhibition of the enzyme in intact cells by both oxalate and pyruvate was found to be similar to that of the purified enzyme. The activity of the enzyme in intact cells indicates that the free solution NAD+ + NADH concentration in erythrocytes is about 10 microM whereas the total extractable NAD+ + NADH is about 80 nmol/ml of cell water.

A p.m.r. isotope-exchange method for studying the kinetic properties of dehydrogenases in intact cells.

A method to determine the activity of dehydrogenases in an intact-cell system is described. The method involves the use of n.m.r. to monitor bulk isotope exchange. The approach is illustrated by application to the isotope equilibration of pyruvate and lactate as catalyzed by lactate dehydrogenase in intact erythrocytes. Particular problems peculiar to bulk isotope exchange and its observation by n.m.r. are considered.

A 1H n.m.r. study of isotope exchange catalysed by glycolytic enzymes in the human erythrocyte.

The exchange of hydrogen and deuterium atoms between the C-2 position of lactate and solvent was monitored in suspensions of human erythrocytes by using a non-invasive spin-echo p.m.r. method that permits continuous assessment of the rate and the extent of exchange. Exchange rates were measured in cells suspended in buffers made in 2H2O and 1H2O after the addition of L-[2-1H]lactate and L-[2-2H]lactate respectively. The rate of exchange is dependent on the activities of four glycolytic enzymes (fructose bisphosphate aldolase, triose phosphate isomerase, glyceraldehyde phosphate dehydrogenase and lactate dehydrogenase) and on the concentrations of their substrates. The dependence of the exchange on the following substrates was studied: (1) lactate, (2) the triose phosphates and fructose 1,6-bisphosphate and (3) pyruvate. Observation of the exchange in vitro, in a system produced by mixing the isolated enzymes, permits determination of the individual isotope-exchange equilibrium velocities of the enzymes. The dependence of the equilibrium velocity of human erythrocyte lactate dehydrogenase on NAD+ + NADH concentration was measured. Possible applications of these methods are discussed.

Studies of pyruvate-water isotope exchange catalysed by erythrocytes and proteins.

Erythrocyte suspensions in buffer made with 2H2O catalyse the exchange of pyruvate protons. This process can be easly observed by spin-echo proton magnetic resonance. The dominant exchange process is shown to be due to the formation of Schiff-base links between pyruvate and amino groups of haemoglobin. Other proteins with free alpha-amino groups also catalyse the exchange. The pH*-dependence of the exchange rate due to hen-egg-white-lysozyme reflects the dissociation of the alpha-amino group.

N.m.r. studies of red cells.

Recent n.m.r. studies of intact red cells are described. With 1H n.m.r. the normal high resolution spectra of red cells, even at high fields, are relatively uninformative because the very large number of resonances from the cells merge into a broad envelope. If a simple 90-tau-180 degree spin echo pulse sequence is used, however, many resonances can all be resolved. These include signals from haemoglobin histidines, glutathione, lactate and pyruvate. 13C and 31P signals have also been seen with a spectrometer converted to observe these nuclei essentially simultaneously. N.m.r. is well suited to monitor the time course of events after a perturbation of the cell system. Lactate increase, glutathione recovery after oxidation and alkylation of glutathione by iodoacetate can all be observed directly in red cell suspensions by means of 1H spin echo n.m.r. This method has also been used to measure isotope exchange (1H-2H) of lactate and of pyruvate at both the C-3 and the C-2 positions, and some of these exchange rates can be interpreted in terms of the activity of specific enzymes in the cells. 1H spin echo n.m.r. has also been used to obtain information about the transport rates of small molecules into cells. By means of the 13C/31P spectrometer and [13C-1] glucose, the 13C enrichment of 2,3-diphosphoglycerate (2,3-DPG) can be monitored at the same time as the levels of 2,3-DPG, ATP and inorganic phosphate are observed by 31P n.m.r.

Proton nuclear magnetic resonance studies on bovine lutropin, its subunits, and on the alpha subunit of pregnant mare serum gonadotropin. Assignment of histidine resonances in the alpha subunit.

The pK values of the 3 histidine residues in the common alpha subunits of bovine and equine glycoprotein hormones have been determined from titration curves generated from their C-2 proton nuclear magnetic resonances at different pH values. Assignment of resonances to specific histidines is based on a comparison between the two species, which have 1 histidine residue in different positions in their sequences, and of the bovine alpha subunit after removal of its histidine 94 by treatment with carboxypeptidases. In both species, those histidines closest to the COOH terminus titrate with near normal pK values of 6.2. The histidine residue found in the bovine subunit at position 87 titrates with an approximate pK value of 5.4. Histidine 83, adjacent to an oligosaccharide moiety in both species, does not titrate over a pH range of 4.0 to 8.0 and thus appears inaccessible to solvent. Similarly, in bovine lutropin-beta, 1 of 3 histidine residues does not titrate between pH 5.0 and 7.0. In the intact hormone, 2 "nontitratable" histidine residues are found. Changes in the characteristics of the signals, however, preclude unambiguous assignment of these two resonances to the nontitrating histidines in the isolated subunits. It appears that changes in the environment of at least some histidines occur when the subunits combine to yield intact hormone.

Application of spin-echo nuclear magnetic resonance to whole-cell systems. Membrane transport.

A new method for studying membrane transport is presented. High resolution n.m.r. is used to measure the distribution of small molecules between the intracellular and extracellular compartments. The method uses spin-echo techniques and relies on a difference in the magnetic susceptibility of the media inside and outside of cells. It also provides simultaneous information on the metabolic status of the cell. The method is illustrated by a study of alanine and lactate transport in the human erythrocyte.

Volatile anesthetic facilitation of in vitro desensitization of membrane-bound acetylcholine receptor from Torpedo californica.

Incubation of membrane fragments bearing acetylcholine receptors from Torpedo californica under an atmosphere of 3% halothane, 1% chloroform, or 6% diethyl ether greatly facilitates the carbamoylcholine-induced structural transition of the acetylcholine receptor reflected by alterations in the rate of binding of (125)I-labeled alpha-bungarotoxin. The half-time of this ligand-induced conformational change is decreased to 10% of the original value after incubation of the membranes with these volatile anesthetics at or near their clinical concentrations. The synergistic effects observed with the general anesthetics and carbamoylcholine are abolished if the membranes are incubated under a stream of air after exposure to the inhalational agents. The antagonist d-tubocurarine exerts a smaller yet measurable time-dependent effect on the toxin-binding properties of the membrane fragments. Treatment of membranes with general anesthetics facilitates this antagonist-induced conversion of the receptor protein as well. The synergism between ligands and general anesthetics may be due to the disruption by these inhalational agents of interactions at the protein-lipid interface, which may play a significant role in determination of receptor conformation. In addition, if the conformational change induced by carbamoylcholine observed in the snake toxin binding assay corresponds to desensitization of the receptor in vivo, facilitation of this conformational change by volatile anesthetics provides an attractive model for the pharmacological action of these compounds.