Bilateral integration of sensorimotor signals during pedaling.
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Bicycling↗
Biomedical subjects
D A Brown
Publications and source records attributed to D A Brown.
Association of GAP-43 with detergent-resistant membranes requires two palmitoylated cysteine residues.
GAP-43 is an abundant protein in axonal growth cones of developing and regenerating neurons. We found that GAP-43 was enriched in detergent-resistant membranes (DRMs) isolated by Triton X-100 extraction from PC12 pheochromocytoma cells and could be detected in detergent-insoluble plasma membrane remnants after extraction of cells in situ. GAP-43 is palmitoylated at Cys-3 and Cys-4. Mutation of either Cys residue prevented association with DRMs. A hybrid protein containing the first 20 amino acid residues of GAP-43 fused to beta-galactosidase was targeted to DRMs even more efficiently than GAP-43 itself. We conclude that tandem palmitoylated Cys residues can target GAP-43 to DRMs, defining a new signal for DRM targeting. We propose that tandem or closely spaced saturated fatty acyl chains partition into domains or "rafts" in the liquid-ordered phase, or a phase with similar properties, in cell membranes. These rafts are isolated as DRMs after detergent extraction. The brain-specific heterotrimeric G protein Go, which may be regulated by GAP-43 in vitro, was also enriched in DRMs from PC12 cells. Targeting of GAP-43 to rafts may function to facilitate signaling through Go. In addition, raft association may aid in sorting of GAP-43 into axonally directed vesicles in the trans-Golgi network.
Animals↗
Intracellular retention of recombinant GABAB receptors.
gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.
Animals↗
Structure and origin of ordered lipid domains in biological membranes.
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Animals↗
P2Y2 nucleotide receptors expressed heterologously in sympathetic neurons inhibit both N-type Ca2+ and M-type K+ currents.
The P2Y2 receptor is a uridine/adenosine triphosphate (UTP/ATP)-sensitive G-protein-linked nucleotide receptor that previously has been reported to stimulate the phosphoinositide signaling pathway. Messenger RNA for this receptor has been detected in brain tissue. We have investigated the coupling of the molecularly defined rat P2Y2 receptor to neuronal N-type Ca2+ channels and to M-type K+ channels by heterologous expression in rat superior cervical sympathetic (SCG) neurons. After the injection of P2Y2 cRNA, UTP inhibited the currents carried by both types of ion channel. As previously reported [Filippov AK, Webb TE, Barnard EA, Brown DA (1997) Inhibition by heterologously expressed P2Y2 nuerones. Br J Pharmacol 121:849-851], UTP inhibited the Ca2+ current (ICa(N)) by up to 64%, with an IC50 of approximately 0.5 microM. We now find that UTP also inhibited the K+M current (IK(M)) by up to 61%, with an IC50 of approximately 1.5 microM. UTP had no effect on either current in neurons not injected with P2Y2 cRNA. Structure-activity relations for the inhibition of ICa(N) and IK(M) in P2Y2 cRNA-injected neurons were similar, with UTP >/= ATP > ITP >> GTP,UDP. However, coupling to these two channels involved different G-proteins: pretreatment with Pertussis toxin (PTX) did not affect UTP-induced inhibition of IK(M) but reduced inhibition of ICa(N) by approximately 60% and abolished the voltage-dependent component of this inhibition. In unclamped neurons, UTP greatly facilitated depolarization-induced action potential discharges. Thus, the single P2Y2 receptor can couple to at least two G-proteins to inhibit both Ca2+N and K+M channels with near-equal facility. This implies that the P2Y2 receptor may induce a broad range of effector responses in the nervous system.
Animals↗
The alpha subunit of Gq contributes to muscarinic inhibition of the M-type potassium current in sympathetic neurons.
Rat superior cervical ganglion (SCG) neurons express low-threshold noninactivating M-type potassium channels (IK(M)), which can be inhibited by activation of M1 muscarinic receptors. This inhibition occurs via pertussis toxin-insensitive G-proteins belonging to the Galphaq family (Caulfield et al., 1994 ). We have used DNA plasmids encoding antisense sequences against the 3' untranslated regions of Galpha subunits (antisense plasmids) to investigate the specific G-protein subunits involved in muscarinic inhibition of IK(M). These antisense plasmids specifically reduced levels of the target G-protein 48 hr after intranuclear injection. In cells depleted of Galphaq, muscarinic inhibition of IK(M) was attenuated compared both with uninjected neurons and with neurons injected with an inappropriate GalphaoA antisense plasmid. In contrast, depletion of Galpha11 protein did not alter IK(M) inhibition. To determine whether the alpha or beta gamma subunits of the G-protein mediated this inhibition, we have overexpressed the C terminus of beta adrenergic receptor kinase 1 (betaARK1), which binds free beta gamma subunits. betaARK1 did not reduce muscarinic inhibition of IK(M) at a concentration of plasmid that can reduce beta gamma-mediated inhibition of calcium current (). Also, expression of beta1gamma2 dimers did not alter the IK(M) density in SCG neurons. In contrast, IK(M) was virtually abolished in cells expressing GTPase-deficient, constitutively active forms of Galphaq and Galpha11. These data suggest that Galphaq is the principal mediator of muscarinic IK(M) inhibition in rat SCG neurons and that this more likely results from an effect of the alpha subunit than the beta gamma subunits of the Gq heterotrimer.
Animals↗
On the role of endogenous G-protein beta gamma subunits in N-type Ca2+ current inhibition by neurotransmitters in rat sympathetic neurones.
1. Using whole-cell and perforated-patch recordings, we have examined the part played by endogenous G-protein beta gamma subunits in neurotransmitter-mediated inhibition of N-type Ca2+ channel current (ICa) in dissociated rat superior cervical sympathetic neurones. 2. Expression of the C-terminus domain of beta-adrenergic receptor kinase 1 (beta ARK1), which contains the consensus motif (QXXER) for binding G beta gamma, reduced the fast (pertussis toxin (PTX)-sensitive) and voltage-dependent inhibition of ICa by noradrenaline and somatostatin, but not the slow (PTX-insensitive) and voltage-independent inhibition induced by angiotensin II. beta ARK1 peptide reduced GTP-gamma-S-induced voltage-dependent and PTX-sensitive inhibition of ICa but not GTP-gamma-S-mediated voltage-independent inhibition. 3. Overexpression of G beta 1 gamma 2, which mimicked the voltage-dependent inhibition by reducing ICa density and enhancing basal facilitation, occluded the voltage-dependent noradrenaline- and somatostatin-mediated inhibitions but not the inhibition mediated by angiotensin II. 4. Co-expression of the C-terminus of beta ARK1 with beta 1 and gamma 2 subunits prevented the effects of G beta gamma dimers on basal Ca2+ channel behaviour in a manner consistent with the sequestering of G beta gamma. 5. The expression of the C-terminus of beta ARK1 slowed down reinhibition kinetics of ICa following conditioning depolarizations and induced long-lasting facilitation by cumulatively sequestering beta gamma subunits. 6. Our findings identify endogenous G beta gamma as the mediator of the voltage-dependent, PTX-sensitive inhibition of ICa induced by both noradrenaline and somatostatin but not the voltage-independent. PTX-insensitive inhibition by angiotensin II. They also support the view that voltage-dependent inhibition results from a direct G beta gamma-Ca2+ channel interaction.
Angiotensin II↗
Cholesterol and sphingolipid enhance the Triton X-100 insolubility of glycosylphosphatidylinositol-anchored proteins by promoting the formation of detergent-insoluble ordered membrane domains.
Glycosylphosphatidylinositol (GPI)-anchored proteins can be isolated from both cells and sphingolipid and cholesterol-rich liposomes (SCRLs) in association with detergent-insoluble membranes. We found previously that detergent insolubility of lipids was characteristic of phases in which lipid acyl chains are ordered. We presented evidence that GPI-anchored proteins are insoluble because they associate with cholesterol and sphingolipid-rich lipid domains with properties similar to the liquid-ordered phase. Here, this model was tested by a variety of approaches. First, we demonstrated that saponin, which removes cholesterol from cell membranes and allows solubilization of GPI-anchored proteins by Triton X-100, had the same effect on the GPI-anchored protein alkaline phosphatase (PLAP) in SCRLs of appropriate lipid composition. The similarity of saponin action in cells and simple liposomes suggests that the compound disrupts protein-lipid interactions. However, direct interactions between PLAP and cholesterol were not needed for insolubility, because the protein was also insoluble in cholesterol-free liposomes containing lipid in an ordered phase. Instead, cholesterol acted by greatly enhancing the formation of a detergent-insoluble phase by sphingolipids, which have a tendency to form ordered phases. We propose that saponin solubilizes GPI-anchored proteins because the lipid composition of cell membranes (and the SCRLs used above) supports ordered phase formation in the presence but not the absence of cholesterol. Supporting this model, saponin did not promote Triton X-100 solubilization of PLAP in SCRLs with sphingolipid levels high enough to allow ordered phase formation in the absence of cholesterol. We also showed that two additional GPI-anchored proteins are detergent-insoluble in SCRLs and that detergent does not artifactually create ordered domains or cause components of solubilized membranes to associate with detergent-resistant membranes present in separate bilayers in the same lysate. We conclude that the ordered domain model explains the behavior of detergent-resistant membranes in liposomes and cells.
Animals↗
Normative data for strength and flexibility of women throughout life.
Previously established normative data for muscular strength, endurance and the flexibility of women have recently been criticized as being unreliable. Furthermore, no normative data for the muscular fitness of women over 70 years of age are established. The purpose of this study was to derive normative data for muscular fitness in women 20-70+ years old, and to compare these data to the most recently published norms of the American College of Sports Medicine (ACSM). A total of 304 independent-living women were evaluated for maximal performance of bench press, leg press, modified push-ups, grip strength, and sit-and-reach. Tables of normative values for each measurement were generated along with percentile rankings. Large discrepancies were found between these new data and those of the ACSM norms. Bench press norms from ACSM are comparatively high so that the average woman in this current sample scored below the 10th percentile of the ACSM rankings. Similarly, the average woman in our sample ranked in either the poor or fair category of the ACSM norms for modified push-ups, and in the poor ACSM category for sit-and-reach. In contrast, leg press scores for the average woman in this study fell within the good or excellent category of the ACSM norms, suggesting that the ACSM norms are too low. Grip strength for the middle-aged women in this study was higher than that in previous norms. These newly established norms are better suited for interpreting women's fitness test results than previously published norms.
Adult↗
Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones.
Small (SKCa) Ca2+-activated K+ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K+ extracellular solution ([K+]o=2.5 mM), SKCa channels had a low conductance (@3 pS at 0 mV), were activated by >/=175 nM Ca2+ (Po=0.54 at 500 nM Ca2+) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SKCa channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SKCa channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones.
Animals↗
G-proteins and G-protein subunits mediating cholinergic inhibition of N-type calcium currents in sympathetic neurons.
One postsynaptic action of the transmitter acetylcholine in sympathetic ganglia is to inhibit somatic N-type Ca2+ currents: this reduces Ca2+-activated K+ currents and facilitates high-frequency spiking. Previous experiments on rat superior cervical ganglion neurons have revealed two distinct pathways for this inhibitory action: a rapid, voltage-dependent inhibition through activation of M4 muscarinic acetylcholine receptors (mAChRs), and a slower, voltage-independent inhibition via M1 mAChRs [Hille (1994) Trends in Neurosci., 17, 531-536]. We have analysed the mechanistic basis for this divergence at the level of the individual G-proteins and their alpha and betagamma subunits, using a combination of site-directed antibody injection, plasmid-driven antisense RNA expression, overexpression of selected constitutively active subunits, and antagonism of endogenously liberated betagamma subunits by over-expression of Dy-binding P-adrenergic receptor kinase 1 (PARK1) peptide. The results indicate that: (i) M4 mAChR-induced inhibition is mediated by GoA; (ii) a and Py subunits released from the activated GoA heterotrimer produce separate voltage-insensitive and voltage-sensitive components of inhibition, respectively; and (iii) voltage-insensitive M1 mAChR-induced inhibition is likely to be mediated by the alpha subunit of Gq. Hence, Ca2+ current inhibition results from the concerted, but independent actions of three different G-protein subunits.
Animals↗
Aliphatic and alicyclic diols induce melanogenesis in cultured cells and guinea pig skin.
We have found that several aliphatic and alicyclic diols induce melanogenesis in cultured S91 mouse melanoma cells and normal human epidermal melanocytes (NHEM). In addition, these compounds induce melanogenesis when applied to guinea pig skin, with transfer of melanin to keratinocytes and formation of "supranuclear caps," as occurs in naturally pigmented skin. The relative order of potency of some of these diols in NHEM is 5-norbornene-2,2-dimethanol > 3,3-dimethyl-1,2-butanediol > cis-1,2-cyclopentanediol > 2,3-dimethyl-2,3-butanediol > 1,2-propanediol. Following treatment with these diols or 3-isobutyl-1-methylxanthine, melanin and tyrosinase activity are increased within S91 cells and NHEM; however, for cultured NHEM, the largest increases of melanin and tyrosinase occur in an extracellular particulate fraction, shown by electron microscopy to consist almost entirely of stage III and IV melanosomes. These results indicate that cultured NHEM treated with diols export melanosomes in a fashion that is commensurate with natural melanogenic processes. In contrast, S91 mouse melanoma cells exhibit aberrant melanosomal trafficking, in accordance with the known defect in myosin-V mediated melanosomal transport. Both S91 cells and NHEM exhibit morphologic changes and growth arrest indicative of differentiation following treatment with diols. The diols described in this report are candidates for use as cosmeceutical tanning agents.
Animals↗
Relationships between timing of muscle excitation and impaired motor performance during cyclical lower extremity movement in post-stroke hemiplegia.
We used an ergometer pedalling paradigm to relate abnormalities in the timing of muscle excitation to the impaired ability to perform mechanical work in the plegic lower limbs of persons with hemiplegia. The EMGs of seven leg muscles and pedal forces were measured bilaterally during pedalling for 15 persons with hemiplegia and 12 neurologically intact age-matched control subjects. Subjects were asked to pedal at a moderate workload (135 J) and cadence (40 r.p.m.). While intersubject variability was high, the external mechanical work output of the plegic leg was significantly less (from 79.6% to -28.9% of the work produced by average leg of control subjects) as a result of less positive work and more negative work being done. The timing of EMG in individual plegic limb muscles exhibited two distinct types of abnormalities that were significantly correlated with this lesser work production: prolonged excitation in the vastus medialis and phase-advanced excitation (both early initiation and early termination) in the rectus femoris and semimembranosus. These results suggest that muscles were differently affected depending on their function, external power-producing muscles (e.g. vastus medialis) showing prolonged excitation and muscles that normally maintain crank progression during limb transitions (e.g. semimembranosus) showing phase-advanced excitation.
Aged↗
Performance following ability-focused physical therapy intervention in individuals with severely limited physical and cognitive abilities.
BACKGROUND AND PURPOSE: Do individuals with severely limited physical and cognitive abilities improve their gross motor abilities when given physical therapy intervention, and does improvement transfer to nontreatment settings? SUBJECTS: The subjects were 24 individuals (10 female, 14 male), aged 3 to 30 years (X = 20.1, SD = 8.1), who were nonambulatory and had limited adaptive behavior. METHODS: Change in gross motor ability during 18 weeks of twice-weekly therapy was measured using goal attainment scaling (GAS). Three gross motor goals were developed for each subject based on individual or caregiver needs, with one goal randomly selected as a control. Physical impairments were treated, and behavioral management principles, low-level communication approaches, high-repetition practice of goals, and a progressive reduction of both physical assistance and multisensory cues were used. An independent rater scored goal level from randomly ordered videotapes recorded during therapy and in recess and home settings. RESULTS: Mean GAS T scores were higher for treatment goals (X = 45.6, SD = 10.5) compared with control goals (X = 34.6, SD = 11.8). When the expected goal level (50) was met during therapy, mean GAS T scores in recess settings ( X = 35.9, SD = 11.5) and home settings (X = 42.2, SD = 12.2) were lower. At the conclusion of therapy, there were no differences in goal levels between treatment and control goals in both the recess and home settings. CONCLUSION AND DISCUSSION: The subjects demonstrated improvement of gross motor abilities practiced during therapy. Level of ability during therapy, however, did not consistently transfer to the recess of home settings. [Brown DA, Effgen SK, Palisano RJ. Performance following ability-focused physical therapy intervention in individuals with severely limited physical and cognitive abilities.
Adolescent↗
Gender effect on beta-endorphin response to exercise.
PURPOSE: Twelve healthy men (26.4 yr) and women (26.8 yr) were compared at rest and after cycling for 25 min at 60 and 80% VO2max to determine whether gender and menstrual cycle influenced circulating beta-endorphin concentration (BE). METHODS: VO2max was determined on a cycle ergometer, and subjects completed the exercise in a randomized order. Women were tested in both the luteal (L) and follicular (F) phases of their menstrual cycle, which was confirmed by their blood estrogen levels. All tests were conducted in the morning after a 30-min rest (12-h postabsorptive). An indwelling venous catheter placed in a forearm vein enabled blood sampling at rest, 25 min of cycling, and 25 min of recovery. RESULTS: Resting BE was similar for men before both 60 and 80% intensities of exercise, 5.27 +/- 0.43 and 5.30 +/- 0.33 pmol.mL-1, respectively. BE was not significantly changed at 60% VO2max (6.54 +/- 0.33 pmol.mL-1) but significantly increased at 80% VO2max (11.90 +/- 1.98 pmol.mL-1). Women tended to have slightly lower BE during the L compared with F, but this did not reach significance (L = 4.40 +/- 0.22, F = 4.73 +/- 0.30 pmol.mL-1). Cycling at 60% VO2max did not significantly increase BE in the L (5.41 +/- 0.42 pmol.mL-1) nor the F (5.35 +/- 0.40 pmol.mL-1). Cycling at 80% VO2max increased BE to a similar extent in both the L and F phase, respectively (10.44 and 10.96). Although the BE concentrations tended to be slightly lower in women compared with men at 80% VO2max, this did not reach statistical significance. CONCLUSIONS: These data suggest that women cycling at 80% VO2max will have a similar BE response to men independent of their menstrual cycle. BE in women at rest and who exercise at lower exercise intensities may have slightly lower BE levels then men independent of the time of the women's menstrual cycle.
Adolescent↗
Functions of lipid rafts in biological membranes.
Recent studies showing that detergent-resistant membrane fragments can be isolated from cells suggest that biological membranes are not always in a liquid-crystalline phase. Instead, sphingolipid and cholesterol-rich membranes such as plasma membranes appear to exist, at least partially, in the liquid-ordered phase or a phase with similar properties. Sphingolipid and cholesterol-rich domains may exist as phase-separated "rafts" in the membrane. We discuss the relationship between detergent-resistant membranes, rafts, caveolae, and low-density plasma membrane fragments. We also discuss possible functions of lipid rafts in membranes. Signal transduction through the high-affinity receptor for IgE on basophils, and possibly through related receptors on other hematopoietic cells, appears to be enhanced by association with rafts. Raft association may also aid in signaling through proteins anchored by glycosylphosphatidylinositol, particularly in hematopoietic cells and neurons. Rafts may also function in sorting and trafficking through the secretory and endocytic pathways.
Animals↗
Sensorimotor state of the contralateral leg affects ipsilateral muscle coordination of pedaling.
The objective of this study was to determine if independent central pattern generating elements controlling the legs in bipedal and unipedal locomotion is a viable theory for locomotor propulsion in humans. Coordinative coupling of the limbs could then be accomplished through mechanical interactions and ipsilateral feedback control rather than through central interlimb neural pathways. Pedaling was chosen as the locomotor task to study because interlimb mechanics can be significantly altered, as pedaling can be executed with the use of either one leg or two legs (cf. walking) and because the load on the limb can be well-controlled. Subjects pedaled a modified bicycle ergometer in a two-legged (bilateral) and a one-legged (unilateral) pedaling condition. The loading on the leg during unilateral pedaling was designed to be identical to the loading experienced by the leg during bilateral pedaling. This loading was achieved by having a trained human "motor" pedal along with the subject and exert on the opposite crank the torque that the subject's contralateral leg generated in bilateral pedaling. The human "motor" was successful at reproducing each subject's one-leg crank torque. The shape of the motor's torque trajectory was similar to that of subjects, and the amount of work done during extension and flexion was not significantly different. Thus the same muscle coordination pattern would allow subjects to pedal successfully in both the bilateral and unilateral conditions, and the afferent signals from the pedaling leg could be the same for both conditions. Although the overall work done by each leg did not change, an 86% decrease in retarding (negative) crank torque during limb flexion was measured in all 11 subjects during the unilateral condition. This corresponded to an increase in integrated electromyography of tibialis anterior (70%), rectus femoris (43%), and biceps femoris (59%) during flexion. Even given visual torque feedback in the unilateral condition, subjects still showed a 33% decrease in negative torque during flexion. These results are consistent with the existence of an inhibitory pathway from elements controlling extension onto contralateral flexion elements, with the pathway operating during two-legged pedaling but not during one-legged pedaling, in which case flexor activity increases. However, this centrally mediated coupling can be overcome with practice, as the human "motor" was able to effectively match the bilateral crank torque after a longer practice regimen. We conclude that the sensorimotor control of a unipedal task is affected by interlimb neural pathways. Thus a task performed unilaterally is not performed with the same muscle coordination utilized in a bipedal condition, even if such coordination would be equally effective in the execution of the unilateral task.
Adult↗
Increased workload enhances force output during pedaling exercise in persons with poststroke hemiplegia.
BACKGROUND AND PURPOSE: A principle of poststroke rehabilitation is that effort should be avoided since it leads to increased spasticity and produces widespread associated abnormal reactions. Although weakness also contributes to movement dysfunction after a stroke, it has been feared that heightened activity levels during strength training will further exacerbate the abnormal tone imbalance present in spastic hemiplegia. The purpose of this study was to test this hypothesis by quantifying the effects of increased workload on motor performance during different speeds of pedaling exercise in persons with poststroke hemiplegia. METHODS: Twelve healthy elderly subjects and 15 subjects with poststroke hemiplegia of greater than 6 months since onset were tested. The experimental protocol consisted of having subjects pedal at 12 randomly ordered workload and cadence combinations (45-J, 90-J, 135-J, and 180-J workloads at 25, 40, and 55 rpm). Pedal reaction forces were measured and used to calculate work done by each leg, including net positive and negative components. An electromyogram was recorded from seven leg muscles. RESULTS: The main finding was that net mechanical work done by the plegic leg increased as workload increased in 75 of 81 instances without increasing the percentage of inappropriate muscle activity. CONCLUSIONS: This study provides evidence that persons with hemiplegia increase force output by their plegic limb when pedaling against higher workloads without exacerbation of impaired motor control. Therefore, exertional pedaling exercise is a beneficial intervention for achieving gains in muscular force output without worsening motor control impairments.
Aged↗