Characterization of the glyoxalase I gene from the vascular wilt fungus Verticillium dahliae.

A glyoxalase I gene homologue (VdGLO1) was identified in the vascular wilt fungus Verticillium dahliae by sequence tag analysis of genes expressed during resting structure development. The results of the current study show that the gene encodes a putative 345 amino acid protein with high similarity to glyoxalase I, which produces S-D-lactoylglutathione from the toxic metabolic by-product methylglyoxal (MG). Disruption of the V. dahliae gene by Agrobacterium tumefaciens-mediated transformation resulted in enhanced sensitivity to MG. Mycelial growth of disruption mutants was severely reduced in the presence of 5 mmol/L MG. In contrast, spore production in liquid medium was abolished at 1 mmol/L MG, although not at physiologically relevant concentrations of

Lactate threshold responses to a season of professional British youth soccer.

OBJECTIVE: To examine the changes in aerobic endurance performance of professional youth soccer players throughout the soccer season. METHODS: Nine youth soccer players were tested at six different time points throughout the soccer season by sub-maximal blood lactate assessment, using an incremental treadmill protocol. Whole blood lactate concentration and heart frequency (Hf) were determined at each exercise stage. Running velocities at the first lactate inflection point (v-T(lac)) and at a blood lactate concentration of 4 mmol l(-1) (v-4mM) were determined. RESULTS: Running velocity at the two lactate thresholds increased from the start of pre-season training to the early weeks of the competitive season, from 11.67 (0.29) to 12.96 (0.28) km h(-1) for v-T(lac), and from 13.62 (0.25) to 14.67 (0.24) km h(-1) for v-4mM (p<0.001). However, v-T(lac )and v-4mM when expressed relative to maximum heart frequency (Hf(max)) remained unchanged. The Hf to blood lactate concentration relationship was unchanged after the pre-season training period. The two expressions of lactate threshold did not reveal differences between each other. CONCLUSION: Running velocity at v-T(lac )and v-4mM increased significantly over the pre-season period, but v-T(lac )and v-4mM were unchanged when expressed relative to Hf(max). This finding may indicate that increased endurance performance may be mainly attributable to alterations in Vo(2max). Although lactate assessment of soccer players is useful for determining endurance training adaptations in soccer players, additional assessment of the other two determinants of endurance performance (Vo(2max) and running economy) may provide more useful information for determining physiological adaptations resulting from soccer training and training interventions.

The effect of ball carrying method on sprint speed in rugby union football players.

Different methods of ball carrying can be used when a player runs with the ball in rugby union. We examined how three methods of ball carrying influenced sprinting speed: using both hands, under the left arm and under the right arm. These methods were compared with running without the ball. Our aim was to determine which method of ball carrying optimizes sprinting speed. Altogether, 48 rugby union players (age 21 +/- 2 years, height 1.83 +/- 0.1 m, body mass 85.3 +/- 12 kg, body fat 14 +/- 5%; mean +/- s) were recruited. The players performed twelve 30-m sprints in total (each player performed three trials under each of three methods of carrying the ball and sprinting without the ball). The design of the study was a form of Latin rectangle, balanced across the trial order for each of the methods and for pairwise combinations of the methods in blocks of four per trial. Each sprint consisted of a 10-m rolling start, followed by a 20-m timed section using electronic timing gates. Compared with sprinting 20 m without the ball (2.58 +/- 0.16 s), using both hands (2.62 +/- 0.16 s) led to a significantly slower time (P < 0.05). Sprinting 20 m with the ball under the left arm (2.61 +/- 0.15 s) or under the right arm (2.60 +/- 0.17 s) was significantly quicker than when using 'both hands' (P < 0.05), and both these methods were significantly slower than when running without the ball (P < 0.05). Accordingly, running with the ball in both hands led to the greatest decrement in sprinting performance, although carrying the ball under one arm also reduced the players' sprinting ability. Our results indicate that to gain a speed advantage players should carry the ball under one arm.

Randomized controlled trial of quadriceps training after proximal femoral fracture.

OBJECTIVE: To determine whether systematic progressive high-intensity quadriceps training increases leg extensor power and reduces disability in patients rehabilitating after proximal femoral fracture. DESIGN: Open parallel group randomized controlled trial comparing the addition of six weeks quadriceps training (40 patients) with standard physiotherapy alone (40 patients). The training group exercised twice weekly, with six sets of 12 repetitions of knee extension (both legs), progressing up to 80% of their one-repetition maximum. SETTING: Orthogeriatric unit, and community follow-up. SUBJECTS: Eighty patients rehabilitating after proximal femoral fracture. MAIN OUTCOME MEASURES: Measurements of leg extensor power (Nottingham Power Rig), functional mobility (elderly mobility score), disability (Barthel Index) and quality of life (Nottingham Health Profile) were made at baseline, after six weeks (at the end of the intervention) and at 16 weeks. RESULTS: Leg extensor power increased significantly in the quadriceps training group (fractured leg mean improvement at six weeks 157% (standard error 16), nonfractured leg 80% (12)) compared with the control group (63% (11) and 26% (8) respectively, unpaired Student's t-test p = 0.007 and p = 0.01 for between-group comparisons). Significant benefits were maintained at 16 weeks. Quadriceps training resulted in a greater increase in elderly mobility scale score compared with standard rehabilitation (between-group difference of 2.5 (95% CI 1.1,3.8) at week 6 and 1.9 (0.4,3.4) at week 16). Barthel score increased significantly from week 0 to 6 in the quadriceps training group compared with controls (Mann-Whitney U-test p = 0.05). Patients in the quadriceps training group scored significantly better in the energy subscore of the Nottingham Health Profile at the end of follow-up (Mann-Whitney U-test p = 0.0185). CONCLUSIONS: Progressive high-intensity quadriceps training in elderly proximal femoral fracture patients increased leg extensor power and reduced disability. This was accompanied by an increase in energy as measured by the Nottingham Health Profile. This intervention may provide a simple practical way of improving outcome in these patients.

Validation of an analytic method of calculating cerebral glucose metabolism using PET.

UNLABELLED: Quantitative modeling of cerebral metabolic rate for glucose (CMRglc) using PET with the FDG method requires calculation of the integral of the time course of radioactivity in arterial plasma. Numeric integration has typically been used but requires 30 or more blood samples taken between 15 s and 100 min after injection of the radiotracer. Our laboratory has developed an alternative integration method that fits the values of the plasma samples to an analytically integrable function using only 4-6 samples taken between 40 and 110 min after radiotracer injection. METHODS: The plasma integrals were calculated by both the analytic and the numeric methods with data from FDG PET studies that were not used in the development of the analytic method. In 39 PET studies from 22 healthy volunteers, 30 plasma samples were taken over 110 min. RESULTS: The plasma integrals determined by the analytic and numeric methods yielded a within-subject correlation coefficient of >0.95 and differences of <10%. CONCLUSION: Because the analytic method requires less blood sampling and does not require sampling immediately after radiotracer injection, the experimental procedure is simplified without loss of accuracy in CMRglc computation, and the effect of missing or incorrect samples is reduced.

Loss of D2 receptor binding with age in rhesus monkeys: importance of correction for differences in striatal size.

The relation between striatal dopamine D2 receptor binding and aging was investigated in rhesus monkeys with PET. Monkeys (n = 18, 39 to 360 months of age) were scanned with 11C-raclopride; binding potential in the striatum was estimated graphically. Because our magnetic resonance imaging analysis revealed a concomitant relation between size of striatum and age, the dynamic positron emission tomography (PET) data were corrected for possible partial volume (PV) artifacts before parameter estimation. The age-related decline in binding potential was 1% per year and was smaller than the apparent effect if the age-related change in size was ignored. This is the first in vivo demonstration of a decline in dopamine receptor binding in nonhuman primates. The rate of decline in binding potential is consistent with in vitro findings in monkeys but smaller than what has been measured previously in humans using PET. Previous PET studies in humans, however, have not corrected for PV error, although a decline in striatal size with age has been demonstrated. The results of this study suggest that PV correction must be applied to PET data to accurately detect small changes in receptor binding that may occur in parallel with structural changes in the brain.

Locus coeruleus neurons from morphine-treated rats do not show opiate-withdrawal hyperactivity in vitro.

In vitro studies have not consistently demonstrated naloxone-precipitated opiate-withdrawal hyperactivity of locus coeruleus neurons. The reason for this inconsistency may be because partial or complete withdrawal occurred during preparation of the locus coeruleus slice. The aim of the present study was to assay opiate withdrawal-related hyperactivity in neurons recorded from locus coeruleus slices while ensuring the maintenance of dependence until naloxone-precipitated withdrawal. Extracellular recordings were obtained from individual locus coeruleus neurons in slices from morphine-treated and drug-naive rats. Morphine 1 microM was present in all solutions during preparation and recording in slices from morphine-treated rats. The average firing rate of the drug-naive controls was 0.93 Hz (+/-0.04 Hz). Bath application of morphine (1 microM) almost completely suppressed firing in drug-naive controls (0.058 Hz, +/-0.04 Hz, n=12), whereas in solutions containing 1 microM morphine, the firing rate of cells from morphine-treated rats averaged 0.71 Hz (+/-0.05 Hz), indicating considerable, but incomplete tolerance. In the same slices, naloxone increased the average spontaneous firing of locus coeruleus cells to 0.96 Hz (+/-0. 04 Hz). Thus, naloxone did not produce withdrawal hyperactivity, but returned the cells from morphine-treated rats to control rates. We conclude that locus coeruleus cells in locus coeruleus slice preparations from morphine-treated rats did not demonstrate withdrawal-related hyperactivity even when dependence was maintained until naloxone-precipitated withdrawal. Thus, our results do not support a role for adaptations intrinsic to locus coeruleus neurons in withdrawal hyperexcitability, but instead imply the necessity of functional afferent activity.

Investigation of subjects with abnormal iron studies: role of the hepatic iron index.

AIM: Genetic haemochromatosis is a common disorder resulting in increased iron deposition in the liver and other organs but can be difficult to diagnose. The aim of this study was to assess the diagnostic value of the conventional tests for iron overload (percentage saturation of transferrin, serum ferritin and grading of iron staining on liver biopsy) and compare these with the newer quantitative biochemical measurements of liver iron. METHOD: A retrospective analysis was made of 108 consecutive patients referred for quantitative liver iron measurements. Iron studies were obtained in 66 of the 108 subjects of whom 60 had abnormal screening tests defined as percent saturation of transferrin (> 60%) and/or ferritin > 350 micrograms/L for females and > 450 micrograms/L for males. Based on clinical features, biochemical data and treatment outcome these 60 subjects were classified as either genetic haemochromatosis, nongenetic haemochromatosis or indeterminate. One patient with treated genetic haemochromatosis was excluded from subsequent analysis. RESULTS: Although the serum ferritin (p < 0.002), percentage saturation of transferrin (p < 0.001), histological iron grade (p < 0.0001) were significantly higher in the genetic haemochromatosis than nongenetic haemochromatosis group there was considerable overlap. Similarly for the hepatic iron concentration (HIC) (p < 0.0001) overlap occurred. The hepatic iron index (HIC/age) gave the best separation with only three cases being misclassified. A correlation between the HII and histological iron index (visualised iron score corrected for age) in 15 subjects gave an r value of 0.72. CONCLUSION: Based on this study we feel that in addition to visual grading of iron in liver biopsies, the hepatic iron index is helpful in establishing a diagnosis of genetic haemochromatosis.

Buprenorphine and morphine produce equivalent increases in extracellular single unit activity of dopamine neurons in the ventral tegmental area in vivo.

Buprenorphine is a synthetic opioid proposed as a potential treatment for drug abuse. Although buprenorphine is widely considered to be a partial agonist at opioid receptors, little is known of its electrophysiological effects in the central nervous system. Because buprenorphine has been reported to have limited hedonic effects in humans, and since activation of the dopaminergic system is thought to be critical to the reinforcing effects of drugs, we compared the ability of buprenorphine and morphine to activate dopamine neurons. We report here that buprenorphine and morphine are equally effective in increasing the impulse flow of dopamine cells in the ventral tegmental area. Extracellular single unit activity was recorded from dopaminergic (DA) neurons in the ventral tegmental area (VTA) of chloral hydrate anesthetized rats. Standard physiological and anatomical criteria were used to identify DA neurons. Systemic injection of buprenorphine (5-200 micrograms/kg, i.v.) and morphine (1-10 mg/kg, i.v.) produced equal magnitudes of activation in a similar subset of DA neurons in the VTA (buprenorphine: 173%; morphine: 164%). Unlike morphine, the activation by buprenorphine was not reversed by the opioid antagonist naloxone (50-100 micrograms/kg, i.v.), but this is consistent with the known pharmacodynamics of buprenorphine at opioid receptors. These studies demonstrate that acute administration of buprenorphine has morphine-like effects on the impulse activity of DA neurons. The implications for use of buprenorphine as a clinical treatment for drug abuse are discussed.

Inhibition of nitric oxide (NO) production selectively impairs learning and memory in the rat.

Animals administered the nitric oxide (NO) synthase inhibitor N-w-nitro-L-arginine methyl ester (NAME) for five days exhibited severe deficits in acquisition of a place-navigation learning task. The effect of NAME was selective to place-navigation learning. NAME had no effect on sensorimotor or motivational processes in a related task. These results are consistent with the view that NO participates in learning and execution of memory tasks.

Both phasic sensory stimulation and tonic pharmacological activation increase Fos-like immunoreactivity in the rat locus coeruleus.

Neuronal activation increases levels of Fos protein, the product of the early immediate gene c-fos. Since most studies used stimuli that evoke sustained elevations in activity; the present study examined Fos-like immunoreactivity (Fos-LI) in the nucleus locus coeruleus (LC). Halothane-anesthetized rats were given either footshock to elicit phasic activation of LC neurons or yohimbine (10 mg/kg, i.p.) to induce a tonic increase in firing. Both treatments markedly increased Fos-LI in a subpopulation of LC neurons. These results demonstrate that c-fos induction does not require high tonic levels of neuronal activity and that Fos-LI may underestimate the proportion of LC neurons neurophysiologically activated by a given stimulus and suggest that factors beyond neuronal activity per se contributes to c-fos expression.

Extracellular characteristics of putative cholinergic neurons in the rat laterodorsal tegmental nucleus.

The extracellular electrophysiological properties of neurons in the laterodorsal tegmental nucleus (LDT), a major source of cholinergic afferents to the thalamus, were studied in chloral hydrate-anesthetized rats. A combination of antidromic activation from the thalamus and histological verification of recording sites was used to correlate the identity of extracellular recordings in the rat LDT with cholinergic neurons in that region. All neurons antidromically activated by stimulation of the anteroventral thalamus were histologically verified to be within clusters of cholinergic (NADPH-d-positive) cells in the LDT or in the adjacent nucleus locus coeruleus (LC). The thalamically projecting LDT neurons had a homogeneous neurophysiological profile consisting of long duration action potentials (mean = 2.5 ms), slow conduction velocities (mean = 0.78 m/s), and lengthy chronaxie values (mean = 0.725 ms). The appearance and axonal characteristics of these neurons resembled those of noradrenergic LC neurons, but the two populations exhibited substantially different spontaneous activity patterns and sensory responsiveness. These characteristics may be useful in the preliminary identification of putative cholinergic neurons in vivo, and thereby provide a foundation for exploring the neuropharmacology, afferent modulation, sensory responsiveness and behavioral correlates of the brainstem cholinergic system.

In vitro electrophysiology of neurons in the lateral dorsal tegmental nucleus.

The lateral dorsal tegmental nucleus (LDT) provides ascending cholinergic projections to forebrain structures such as prefrontal cortex, septum, habenula, and thalamus, but relatively little is known of the physiology of LDT neurons. Intracellular recordings from LDT neurons in guinea pig brain slices found that most neurons fired action potentials either tonically or in bursts. The voltage dependent characteristics of the neurons suggest that a prolonged afterhyperpolarization due to an outward potassium current and a low-threshold calcium conductance contributed to these two modes of firing. Intracellular injections of Lucifer Yellow and subsequent staining for NADPH-diaphorase activity permitted positive identification of cholinergic neurons.

Responses of primate locus coeruleus neurons to simple and complex sensory stimuli.

Single and multiple unit recordings were obtained from locus coeruleus (LC) of unanesthetized, chair-restrained monkeys during presentation of a range of sensory stimuli. Tonic activity was higher during alertness or agitation than during behavioral inattentiveness and drowsiness. Low-level, simple auditory stimuli elicited no response, while more intense stimuli evoked phasic discharges in LC activity. The most pronounced responses were elicited by aversive air puffs and by multi-modal naturalistic stimuli such as interactions with the experimenter. The results suggest that sensory stimuli effective in eliciting LC discharge have specific stimulus attributes. It is proposed that the LC is tuned to specifically respond to stimuli which are conspicuous to that species: stimuli which by their physical or behavioral properties evoke a change in the focus of attention. The LC response would thereby contribute to adaptive behavioral responses to such unexpected imperative stimuli. This hypothesis is consistent with earlier suggestions that the LC contributes to behavioral functions such as vigilance and alarm and provides a rigorous framework for future experiments.

Behavior of monkeys during opiate withdrawal and locus coeruleus stimulation.

The noradrenergic nucleus locus coeruleus (LC) has been implicated in morphine withdrawal. The behavioral effects of opiate antagonist-precipitated morphine withdrawal in chair-restrained Macaca arctoides were therefore compared with LC electrical field stimulation. Both continuous LC stimulation and administration of low doses of naloxone to morphine pellet implanted monkeys produced a significant increase in the same group of behaviors reported previously to follow activation of the LC, without significant increases in general activity or distress behaviors. Signs of autonomic hyperactivity and distress were observed at high doses of naloxone, but not during LC stimulation. Monkeys which had not received morphine treatment did not specifically increased during low intensity LC stimulation is also selectively increased during naloxone-precipitated morphine withdrawal. These data are consistent with and suggest a behavioral consequence of the interactions of opioids with the LC reported at the molecular, intracellular, and cellular level.

Clonidine infusions into the locus coeruleus attenuate behavioral and neurochemical changes associated with naloxone-precipitated withdrawal.

Clonidine, an alpha-2-adrenergic agonist, suppresses signs of opiate withdrawal in animals and in man. Electrical or chemical stimulation of the nucleus locus coeruleus (LC) increases noradrenergic activity and brain concentration of the noradrenergic metabolite MHPG, and produces many signs of opiate withdrawal. Thus, clonidine's ability to attenuate withdrawal might be due to the reduction of noradrenergic neuronal activity originating in the LC, but additional alpha-2-adrenergic receptors throughout the body and other mechanisms may also play a role. The present study explored the neuroanatomical and pharmacological selectivity of alpha-2-adrenergic receptors of the LC in the anti-withdrawal action of clonidine. Experiment 1 tested the hypothesis that behavioral and biochemical measures of naloxone-precipitated withdrawal from morphine would be blocked by infusions of clonidine (0.6 or 2.4 micrograms/microliters) into the LC. Significant reductions were observed in the occurrence of diarrhea, ptosis, weight loss and wet-dog shakes. Clonidine also reversed the naloxone-precipitated increase in hippocampus MHPG concentration. In experiment 2 subjects received an LC infusion or IP injection of a non-lipophilic alpha-2-agonist (ST-91), which does not penetrate the blood-brain barrier, or of clonidine into the dorsal parabrachial nucleus (DPB) to test the selectivity of the effects of clonidine infusions into the LC. ST-91 infusions into the LC reduced several of the observed withdrawal signs and increased others (e.g., jumping). Although peripheral injections of ST-91 attenuated some of the checked signs associated with naloxone-precipitated withdrawal, the frequency of wet-dog shakes was not reduced. ST-91 infusions into the LC, but not systemic ST-91 administration, prevented the withdrawal-induced increase in hippocampus MHPG concentration. Clonidine infused lateral to the LC into the DPB did not significantly attenuate withdrawal or reduce hippocampus MHPG levels. These results provide behavioral and biochemical evidence to support the suggestion that clonidine significantly attenuates naloxone-precipitated withdrawal through an interaction with noradrenergic neurons located in the vicinity of the LC.