Effects of ischemic preconditioning on economy, VO2 kinetics and cycling performance in endurance athletes.

The aim of this study was to determine the effect of ischemic preconditioning (IPC) on several measures of aerobic function and 4-km cycling time-trial performance. An acute cross-over design was adopted involving eight well-trained cyclists (age 27.0 ± 7.0 years) who completed incremental and square-wave exercise tests for determination of peak O2 uptake (VO2peak), ventilatory threshold (VT) and moderate- and heavy-intensity domain VO2 kinetics, as well as 4-km time trials. All were preceded by IPC, or sham-IPC, involving repeated bouts of thigh blood flow occlusion, interspersed with reperfusion. There was no significant difference between IPC and sham-IPC with respect to VO2peak (4.4 ± 0.6 L min-1 vs 4.4 ± 0.5 L min-1, effect size - 0.01 ± 0.09), VT (3.4 ± 0.6 L min-1 vs 3.5 ± 0.5 L min-1, effect size 0.07 ± 0.28), cycling economy (4.9 ± 4.9%, ES 0.24 ± - 0.24, P > 0.05) or any moderate-domain VO2 kinetic parameter. During heavy-intensity exercise, a reduced end-exercise VO2, slow component amplitude and overall gain was observed following IPC compared to sham-IPC. Though not statistically significant, there was a possibly beneficial effect of IPC on 4-km time-trial mean power output (2.2 ± 2.0%; effect size: 0.18 ± 0.15, P > 0.05). The observed reduction in VO2 slow component and tendency for improved economy and 4-km time-trial performance, albeit small, suggests that acute IPC shows some potential as a performance-enhancing priming strategy for well-trained cyclists prior to high-intensity exercise.

Potentiation of M1 Muscarinic Receptor Reverses Plasticity Deficits and Negative and Cognitive Symptoms in a Schizophrenia Mouse Model.

Schizophrenia patients exhibit deficits in signaling of the M1 subtype of muscarinic acetylcholine receptor (mAChR) in the prefrontal cortex (PFC) and also display impaired cortical long-term depression (LTD). We report that selective activation of the M1 mAChR subtype induces LTD in PFC and that this response is completely lost after repeated administration of phencyclidine (PCP), a mouse model of schizophrenia. Furthermore, discovery of a novel, systemically active M1 positive allosteric modulator (PAM), VU0453595, allowed us to evaluate the impact of selective potentiation of M1 on induction of LTD and behavioral deficits in PCP-treated mice. Interestingly, VU0453595 fully restored impaired LTD as well as deficits in cognitive function and social interaction in these mice. These results provide critical new insights into synaptic changes that may contribute to behavioral deficits in this mouse model and support a role for selective M1 PAMs as a novel approach for the treatment of schizophrenia.

Game performance and intermittent hypoxic training.

Live high-train low altitude exposure simulated by hypoxic devices may improve athletic performance. In this study, intermittent normobaric hypoxia was achieved with the GO2altitude hypoxicator to determine its effects on sea level performance in rugby players. Ten players were randomly assigned to two groups. Players in each group received 14 sessions of either hypoxic (10-15% O(2)) or normoxic (21% O(2)) exposure at rest over 14 consecutive days in a single blind fashion. Various performance measures were obtained consecutively in a single testing session pre- and post-exposure. Effects of hypoxic exposure on maximum speed and sprint times were trivial (<1.0%) but unclear (90% likely range, +/-5% to +/-9%). In rugby simulation, hypoxic exposure produced impairments of peak power in two scrums (15%, +/-8%; 9%, +/-7%) and impairments of time in offensive sprints (7%, +/-8%) and tackle sprints (11%, +/-9%). Pending further research, rugby players would be unwise to use normobaric intermittent hypoxic exposure to prepare for games at sea level.

Comparison of neuronal and hemodynamic measures of the brain response to visual stimulation: an optical imaging study.

The noninvasive mapping of hemodynamic brain activity has led to significant advances in neuroimaging. This approach is based in part on the assumption that hemodynamic changes are proportional to (and therefore constitute a linear measure of) neuronal activity. We report a study investigating the quantitative relationship between neuronal and hemodynamic measures. This study exploited the fact that optical imaging methods can simultaneously provide noninvasive measures of neuronal and hemodynamic activity from the same region of the brain. We manipulated visual stimulation frequency and measured responses from the medial occipital area of 8 young adults. The results were consistent with a model postulating a linear relationship between the neuronal activity integrated over time and the amplitude of the hemodynamic response. The hemodynamic response colocalized with the neuronal response. These data support the use of quantitative neuroimaging methods to infer the intensity and localization of neuronal activity in occipital areas.

Students' anxiety in a senior thesis course.

The State-Trait Anxiety Inventory was administered on Weeks 8, 12, and 15 of a semester to 16 students enrolled in a senior thesis course. State anxiety scores were elevated when oral presentations began and declined following the presentations. Trait anxiety scores remained constant across test administrations. The influence of situational variables on students' anxiety was discussed.

SRC binding to the cytoskeleton, triggered by growth cone attachment to laminin, is protein tyrosine phosphatase-dependent.

The interaction of the non-receptor tyrosine kinase, Src, with the cytoskeleton of adhesion sites was studied in nerve growth cones isolated from fetal rat brain. Of particular interest was the role of protein tyrosine phosphatases in the regulation of Src-cytoskeleton binding. Growth cones were found to contain a high level of protein tryrosine phosphatase activity, most of it membrane-associated and forming large, multimeric and wheat germ agglutinin-binding complexes. The receptor tyrosine phosphatase PTPalpha seems to be the most prevalent species among the membrane-associated enzymes. As seen by immunofluorescence, PTPalpha is present throughout the plasmalemma of the growth cone including filopodia, and it forms a punctate pattern consistent with that of integrin beta1. For adhesion site analysis, isolated growth cones were either plated onto the neurite growth substratum, laminin, or kept in suspension. Plating growth cones on laminin triggered an 8-fold increase in Src binding to the adherent cytoskeleton. This effect was blocked completely with the protein tyrosine phosphatase inhibitor, vanadate. Growth cone plating also increased the association with adhesion sites of tyrosine phosphatase activity (14-fold) and of PTPalpha immunoreactivity (6-fold). Vanadate blocked the enzyme activity but not the recruitment of PTPalpha to the adhesion sites. In conjunction with our previous results on growth cones, these data suggest that integrin binding to laminin triggers the recruitment of PTPalpha (and perhaps other protein tyrosine phosphatases) to adhesion sites, resulting in de-phosphorylation of Src's tyr 527. As a result Src unfolds, becomes kinase-active, and its SH2 domain can bind to an adhesion site protein. This implies a critical role for protein tyrosine phosphatase activity in the earliest phases of adhesion site assembly.

Targeting and modification of prokaryotic cell-cell junctions by tobacco mosaic virus cell-to-cell movement protein.

The movement protein (MP) of tobacco mosaic virus (TMV) facilitates the cell-to-cell spread of infection by altering the structure and function of plasmodesmata, the intercellular communication channels in plants. Because the protein was shown to interfere with intercellular communication when expressed in the cyanobacterium Anabaena sp. strain PCC 7120, whether the ability of the protein to target and to modify intercellular communication channels in plants is conserved in this prokaryote was investigated. It was found that the MP localizes to the cell junctions and induces the formation of filamentous structures that traverse the septa. It is proposed that the protein interacts with host components that are similar between plants and Anabaena and that may be evolutionarily related. The observations in Anabaena suggest that the MP modifies plasmodesmata by forming a filamentous aggregate within the pore.

Memory-driven processing in human medial occipital cortex: an event-related optical signal (EROS) study.

Memory-driven processing in medial occipital areas (Area V1 and immediately adjacent structures) was investigated noninvasively using the event-related optical signal (EROS). Subjects viewed two letter stimuli presented in the left and right hemifields, respectively. They then viewed a centrally presented test letter and had to indicate whether this letter was the same as either of the letters presented earlier. The initial EROS response to the test stimulus in medial occipital areas (latency: 50-150 ms) was unilaterally suppressed in the hemisphere previously exposed to the same stimulus. This finding suggests that medial occipital cortex activity is modulated by a rapidly adapting hemispheric-specific pattern recognition mechanism.

The arginine taste receptor. Physiology, biochemistry, and immunohistochemistry.

The amino acid, L-arginine (L-Arg), is a potent taste stimulus for the channel catfish, Ictalurus punctatus. Receptor binding studies demonstrated a high-affinity binding of L-Arg to putative taste receptor sites. This binding could be inhibited by preincubation of the tissue in the lectins Phaseolus vulgaris agglutinin (PHA) and Ricinus communis agglutinin I (RCA I). Neurophysiological studies demonstrated that the L-Arg receptor is a stimulus-gated ion channel type receptor whose conductance was stimulated by L-Arg and inhibited by D-arginine (D-Arg). To purify the receptor we subjected CHAPS solubilized partial membrane preparation from barbel epithelium to RCA I lectin affinity chromatography. The bound proteins were eluted with D-galactose. When these proteins were reconstituted into lipid bilayers, L-Arg activated single channel currents with conductances between 45 and 85 pS. Sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the eluted protein showed a distinct band at approximately 83 kDa. Polyclonal antibodies raised against this 83-kDa band in guinea pigs reacted with numerous small (approximately 1 micron) sites within the taste pore of every taste bud when applied to fixed nonpermeabilized barbels. This observation suggests that the antibodies recognize an externally-facing epitope of the putative Arg receptor. The antibodies also inhibited L-Arg-stimulated currents in reconstitution studies. Sephacryl S-300 HR chromatography of the eluant from the affinity column showed a high molecular weight peak (> 700 kDa) which was recognized by the antibodies. Reconstitution of the protein from this peak into a lipid bilayer resulted in L-Arg-stimulated channels that could be inhibited by D-Arg. This high molecular weight component may be aggregates of the arginine taste receptor.

The effect of streptomycin, oxytetracycline, tilmicosin and phenylbutazone on spermatogenesis in bulls.

To determine whether declining semen quality associated with health problems may be due to certain antibiotic or anti-inflammatory treatments, semen was collected 3 times per week for up to 42 d from 6 normal bulls after treatment with oxytetracycline, tilmicosin, dihydrostreptomycin, or phenylbutazone. No adverse effects on semen quality were observed.

Fast and localized event-related optical signals (EROS) in the human occipital cortex: comparisons with the visual evoked potential and fMRI.

Localized evoked activity of the human cortex produces fast changes in optical properties that can be detected noninvasively (event-related optical signal, or EROS). In the present study a fast EROS response (latency approximately 100 ms) elicited in the occipital cortex by visual stimuli showed spatial congruence with fMRI signals and temporal correspondence with VEPs, thus combining subcentimeter spatial localization with subsecond temporal resolution. fMRI signals were recorded from striate and extrastriate cortex. Both areas showed EROS peaks, but at different latencies after stimulation (100 and 200-300 ms, respectively). These results suggest that EROS manifests localized neuronal activity associated with information processing. The temporal resolution and spatial localization of this signal make it a promising tool for studying the time course of activity in localized brain areas and for bridging the gap between electrical and hemodynamic imaging methods.

Characterization of phospholipase A2 activity enriched in the nerve growth cone.

Nerve growth cones isolated from fetal rat brain exhibit in their cytosol a robust level of phospholipase A2 activity hydrolyzing phosphatidylinositol (PI) and phosphatidylethanolamine (PE) but not phosphatidylcholine (PC). Western blot analysis with an antibody to the well-characterized cytosolic phospholipase A2 (mol wt 85,000) reveals only trace amounts of this PC- and PE-selective enzyme in growth cones. By gel filtration on Superose 12, growth cone phospholipase A2 activity elutes essentially as two peaks of high molecular mass, at approximately 65 kDa and at well over 100 kDa. Anion exchange chromatography completely separates a PI-selective from a PE-selective activity, indicating the presence of two different, apparently monoselective phospholipase A2 species. The PI-selective enzyme, the predominant phospholipase A2 activity in whole growth cones, is enriched greatly in these structures relative to their parent fractions from fetal brain. This phospholipase A2 is resistant to reducing agents and is found in the cytosol as well as membrane-associated in the presence of Ca2+. However, its catalytic activity is Ca(2+)-independent regardless of whether the enzyme is associated with pure substrate or mixed-lipid growth cone vesicles. The PE-selective phospholipase A2 in growth cones was studied in less detail but shares with the PI-selective enzyme several properties, including intracellular localization, the existence of cytosolic and membrane-associated forms, and Ca2+ independence. Our data indicate growth cones contain two high-molecular-weight forms of phospholipase A2 that share many properties with known, Ca(2+)-independent cytosolic phospholipase A2 species but that appear to be monoselective for PI and PE, respectively. In particular, the PI-selective enzyme may represent a new member of the growing family of cytoplasmic phospholipases A2. The enrichment of the PI-selective phospholipase A2 in growth cones suggests it plays a major role in the regulation of growth cone function.

Axonal origin and purity of growth cones isolated from fetal rat brain.

The investigation of the molecular properties of nerve growth cones depends to a significant degree on their isolation from fetal brain in the form of 'growth cone particles' (GCPs). The availability of markers for developing axons and dendrites, as well as glial cells, has made it possible to characterize the GCP fraction in much greater detail than before and to optimize its yield. Marker analyses show that a member of the N-CAM family (5B4-CAM), synaptophysin, and especially GAP-43 and non-phosphorylated tau, are enriched in the GCP fraction. In contrast, MAP2 and, particularly, glial fibrillary acidic protein and vimentin are fractionated away from GCPs. Furthermore, GCP yield can be doubled relative to the original procedure, without compromising purity, by raising the sucrose concentration of the fractionation gradient's uppermost layer. The results indicate that GCPs are highly purified growth cone fragments with very little glial contamination, and that they are primarily of axonal origin.

Na+ channel changes in the growth cone and developing nerve terminal.

Previous saxitoxin binding studies indicated two forms of the sodium channel in the fetal rat brain; a low-affinity precursor located in an internal membrane compartment, present exclusively in growth cones and a high-affinity mature form present in the plasmalemma of growth cones and characteristic of synapses. This raises the questions (1) of the presence or absence of the beta2 subunit in these channel forms and (2) of the developmental regulation of the the beta2 subunit. Antibodies against the alpha and beta2 channel subunits were used to probe Western blots of subcellular fractions from rat brains at embryonic day 18 (E18), pups at postnatal (P) days 7-25, and adults, as well as purified sodium channels from adult brain. In both synaptosomes and the purified sodium channel the beta2 antibody recognized the expected band at 38 kDa under reducing conditions. However, in contrast to the alpha subunit, this band was absent at E18 and became apparent only from P7 onwards. At the earlier time intervals a very prominent immunoreactive band of unknown identity was evident at 260--300kDa, which declined in intensity concomitant with the appearance of the 38 kDa beta2 band. These data indicate that beta2 subunits are regulated independently from alpha subunits, are absent in differentiating neurons, and hence are not necessary for insertion of the sodium channel into the plasmalemma, at least during early development of the neuron.

Synthesis and biological activity of novel cephalosporins containing a (Z)-vinyl dimethylphosphonate group.

A series of cephalosporins containing a novel 7-[2-(Z)-(2-amino-thiazol-4- yl)-3-(dimethoxy-phosphoryl)-acryloylamino] group were prepared and their antibacterial activity measured against a range of pathogens. In general the compounds displayed a broad spectrum of activity against both Gram positive and Gram negative organisms, except Pseudomonas aeruginosa. Activity against the latter could be achieved by introducing a catechol moiety at the 3 position of the cephalosporin. The methyl phosphonates in general were stable to a wide range of beta-lactamases, including the TEM enzymes and the Enterobacter cloacae P99 chromosomal enzyme. In addition, they showed the advantage of being highly water soluble.

A chloride channel reconstituted from fetal rat brain growth cones.

Chloride channels were reconstituted into planar lipid bilayers isolated from a preparation of growth cone particles (GCPs) isolated from fetal rat brain. One type of channel was predominantly seen and some of its biophysical and pharmacological properties were studied. The single channel i-V relationship was curvilinear with a chord conductance of 75 pS at +30 mV in symmetric 200 mM NaCl solutions buffered with phosphate. The channel was inactivated by depolarization, and this inactivation was reversed rapidly upon returning to -25 mV. The Cl- channel was significantly permeant to Na+ ions (PNa/PCl = 0.26), and the relative halide permeabilities were determined to be: I(1.92) > Br(1.73) > Cl(1.0) > F(0.34). The channel was inhibited by the common stilbene compounds (DIDS, SITS, DNDS), as well as by Zn2+ ions and an indanyloxyacetic acid derivative. A developmental role for the GCP Cl- channel is suggested by the observation that adult rat brain synaptosomal membranes were nearly devoid of this type of Cl- channel.

Plasmalemmal insertion and modification of sodium channels at the nerve growth cone.

We have characterized voltage-dependent sodium channels in growth cones (GCPs) isolated from fetal rat brain using saxitoxin and TTX binding as well as recordings from channels reconstituted into lipid bilayer membranes. Both high- and low-affinity binding sites are present in GCP membranes. However, the two binding sites are segregated largely or completely, with the high-affinity binding sites in the plasmalemma, and the low-affinity sites in an internal membrane compartment. Plasmalemmal insertion of these internal sites can be triggered by high-potassium depolarization and depends on a metalloendoprotease-requiring mechanism. These observations indicate that a precursor-product relationship exists between the internal and external sodium channels of the growth cone, and therefore suggest that channel externalization causes conversion of low-affinity to high-affinity saxitoxin receptors. This conversion may represent a step of channel capacitation.