Dynamics of motor nerve terminal remodeling unveiled using SNARE-cleaving botulinum toxins: the extent and duration are dictated by the sites of SNAP-25 truncation

Nerve sprouts emerge from motor nerve terminals following blockade of exo-endocytosis for more than 3 days by botulinum neurotoxin (BoNT), and form functional synapses, albeit temporary. Upon restoration of synaptic activity to the parent terminal 7 and 90 days after exposure to BoNT/F or A respectively, a concomitant retraction of the outgrowths was observed. BoNT/E caused short-term neuroparalysis, and dramatically accelerated the recovery of BoNT/A-paralyzed muscle by further truncation of SNAP-25 and its replenishment with functional full-length SNARE. The removal of 9 C-terminal residues from SNAP-25 by BoNT/A leads to persistence of the inhibitory product due to the formation of a nonproductive SNARE complex(es) at release sites, whereas deletion of a further 17 amino acids permits replenishment and a speedy recovery. (C) 2003 Elsevier Science (USA). All rights reserved.

Sensory hypersensitivity occurs soon after whiplash injury and is associated with poor recovery

Hypersensitivity to a variety of sensory Stimuli is a feature of persistent whiplash associated disorders (WAD). However, little is known about sensory disturbances from the time Of injury until transition to either recovery or symptom persistence. Quantitative sensory testing (pressure and thermal pain thresholds, the brachial plexus provocation test), the sympathetic vasoconstrictor reflex and psychological distress (GHQ-28) were prospectively measured in 76 whiplash Subjects within 1 month of injury and then 2, 3 and 6 months post-injury. Subjects were classified at 6 months post-injury using scores on the Neck Disability Index: recovered (30). Sensory and sympathetic nervous system tests were also measured in 20 control subjects. All whiplash groups demonstrated local mechanical hyperalgesia in the cervica spine at 1 month post-injury. This hyperalgesia persisted in those with moderate/severe symptoms at 6 months but resolved by 2 months in those who had recovered or reported persistent mild symptoms. Only those with persistent moderate/severe symptoms at 6 months demonstrated generalised hypersensitivity to all sensory tests. These changes Occurred within 1 month of injury and remained Unchanged throughout the Study period. Whilst no significant group differences were evident for the sympathetic vasoconstrictor response, the moderate/severe group showed a tendency for diminished sympathetic reactivity. GHQ-28 scores of the moderate/severe group were higher than those of the other two groups. The differences in GHQ-28 did not impact on any of the sensory measures. These findings suggest that those with persistent moderate/severe symptoms at 6 months display, soon after injury, generalised hypersensitivity suggestive of changes in central pain processing mechanisms. This phenomenon did not Occur in those who recover or those with persistent mild symptoms. (C) 2003 International Association for the Study of Pain. Published by Elsevier Science B.V. All rights reserved.

Effects of revascularisation and contractile reserve on left ventricular remodelling in patients with impaired left ventricular function

Objective: We sought to define the influence of revascularisation and contractile reserve on left ventricular (LV) remodelling in patients with LV dysfunction after myocardial infarction. Revascularisation of viable myocardium is associated with improved regional function, but the effect on remodelling is undefined. Methods: We studied 70 patients with coronary artery disease and LV dysfunction, 31 of whom underwent revascularisation. A standard dobutamine stress echocardiogram (DbE) was carried out. All patients underwent standard medical treatment; the decision to revascularise was made clinically, independent of this study. LV volumes and ejection fraction were measured by 3D echocardiography at baseline and after an average of 40 weeks. Results: There was no significant difference in baseline ejection fraction or volumes between patients who underwent revascularisation and the remainder. Compared to medically treated patients, revascularised patients had significant improvements in ejection fraction and end-systolic volume in follow-up. The impact of baseline variables on remodelling was assessed by dividing patients into tertiles of LV ejection fraction and volumes. Revascularised patients in the lowest tertile of ejection fraction at baseline (<38%) had a significant improvement in end-systolic volume and ejection fraction, larger than obtained in medically treated patients with low ejection fraction. Revascularised patients with an ejection fraction >38% did not show significant improvement in volumes compared to baseline. Revascularised patients in the largest tertiles of end-systolic (>88 ml) or end-diastolic volume (>149 ml) at baseline had a significant improvement in end-systolic volume. Conclusion: Remodeling appears to occur independent of the presence of regional contractile reserve but does correlate with the volume response to low-dose dobutamine. (C) 2003 Elsevier Ireland Ltd. All rights reserved.

The cytoskeleton and motor proteins of human schistosomes and their roles in surface maintenance and host-parasite interactions

Schistosomes are parasitic blood flukes, responsible for significant human disease in tropical and developing nations. Here we review information on the organization of the cytoskeleton and associated motor proteins of schistosomes, with particular reference to the organization of the syncytial tegument, a unique cellular adaptation of these and other neodermatan flatworms. Extensive EST databases show that the molecular constituents of the cytoskeleton and associated molecular systems are likely to be similar to those of other eukaryotes, although there are potentially some molecules unique to schistosomes and platyhelminths. The biology of some components, particular those contributing to host-parasite interactions as well as chemotherapy and immunotherapy are discussed. Unresolved questions in relation to the structure and function of the tegument relate to dynamic organization of the syncytial layer. (C) 2004 Wiley Periodicals, Inc.

Thirty-year recovery trend in the once depleted Hawaiian green sea turtle stock

The green sea turtle is one of the long-lived species that comprise the charismatic marine megafauna. The green turtle has a long history of human exploitation with some stocks extinct. Here we report on a 30-year study of the nesting abundance of the green turtle stock endemic to the Hawaiian Archipelago. We show that there has been a substantial long-term increase in abundance of this once seriously depleted stock following cessation of harvesting since the 1970s. This population increase has occurred in a far shorter period of time than previously thought possible. There was also a distinct 3-4 year periodicity in annual nesting abundance that might be a function of regional environmental stochasticity that synchronises breeding behaviour throughout the Archipelago. This is one of the few reliable long-term population abundance time series for a large long-lived marine species, which are needed for gaining insights into the recovery process of long-lived marine species and long-term ecological processes. (C) 2003 Elsevier Ltd. All rights reserved.

Reappraising contemporary theories of subcortical participation in language: Proposing an interhemispheric regulatory function for the subthalamic nucleus (STN) in the mediation of high-level linguistic processes

Apropos the basal ganglia, the dominant striatum and globus pallidus internus (GPi) have been hypothesised to represent integral components of subcortical language circuitry. Working subcortical language theories, however, have failed thus far to consider a role for the STN in the mediation of linguistic processes, a structure recently defined as the driving force of basal ganglia output. The aim of this research was to investigate the impact of surgically induced functional inhibition of the STN upon linguistic abilities, within the context of established models of basal ganglia participation in language. Two males with surgically induced 'lesions' of the dominant and non-dominant dorsolateral STN, aimed at relieving Parkinsonian motor symptoms, served as experimental subjects. General and high-level language profiles were compiled for each subject up to 1 month prior to and 3 months following neurosurgery, within the drug-on state (i.e., when optimally medicated). Comparable post-operative alterations in linguistic performance were observed subsequent to surgically induced functional inhibition of the left and right STN. More specifically, higher proportions of reliable decline as opposed to improvement in post-operative performance were demonstrated by both subjects on complex language tasks, hypothesised to entail the interplay of cognitive-linguistic processes. The outcomes of the current research challenge unilateralised models of functional basal ganglia organisation with the proposal of a potential interhemispheric regulatory function for the STN in the mediation of high-level linguistic processes.

Molecular structure and function of the glycine receptor chloride channel

The glycine receptor chloride channel (GlyR) is a member of the nicotinic acetylcholine receptor family of ligand-gated ion channels. Functional receptors of this family comprise five subunits and are important targets for neuroactive drugs. The GlyR is best known for mediating inhibitory neurotransmission in the spinal cord and brain stem, although recent evidence suggests it may also have other physiological roles, including excitatory neurotransmission in embryonic neurons. To date, four alpha-subunits (alpha1 to alpha4) and one beta-subunit have been identified. The differential expression of subunits underlies a diversity in GlyR pharmacology. A developmental switch from alpha2 to alpha1beta is completed by around postnatal day 20 in the rat. The beta-subunit is responsible for anchoring GlyRs to the subsynaptic cytoskeleton via the cytoplasmic protein gephyrin. The last few years have seen a surge in interest in these receptors. Consequently, a wealth of information has recently emerged concerning Glyl? molecular structure and function. Most of the information has been obtained from homomeric alpha1 GlyRs, with the roles of the other subunits receiving relatively little attention. Heritable mutations to human GlyR genes give rise to a rare neurological disorder, hyperekplexia (or startle disease). Similar syndromes also occur in other species. A rapidly growing list of compounds has been shown to exert potent modulatory effects on this receptor. Since GlyRs are involved in motor reflex circuits of the spinal cord and provide inhibitory synapses onto pain sensory neurons, these agents may provide lead compounds for the development of muscle relaxant and peripheral analgesic drugs.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 muM, and inhibition is complete at about 10 muM. In this range, the tubulin assembly is fully ( up to 6 muM) or partially (similar to 6 - 10 muM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 muM Hg2+, the IC50 5.8 muM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay'' mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 muM and a complete inhibition is reached at 1 muM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 muM HgCl2. Between 15 and 20 muM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 muM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations ( 100 muM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Effect of prolonged inactivity on skeletal motor nerve terminals during aestivation in the burrowing frog, Cyclorana alboguttata

This study examined the effect of prolonged inactivity, associated with aestivation, on neuromuscular transmission in the green-striped burrowing frog, Cyclorana alboguttata. We compared the structure and function of the neuromuscular junctions on the iliofibularis muscle from active C. alboguttata and from C. alboguttata that had been aestivating for 6 months. Despite the prolonged period of immobility, there was no significant difference in the shape of the terminals (primary, secondary or tertiary branches) or the length of primary terminal branches between aestivators and non-aestivators. Furthermore, there was no significant difference in the membrane potentials of muscle fibres or in miniature end plate potential (EPP) frequency and amplitude. However, there was a significant decrease in evoked transmitter release characterised by a 56% decrease in mean EPP amplitude, and a 29% increase in the failure rate of nerve terminal action potentials to evoke transmitter release. The impact of this suite of neuromuscular characteristics on the locomotor performance of emergent frogs is discussed.

Borrowing from models of motor control to translate cognitive processes: Evidence for hypokinetic-hyperkinetic linguistic homologues?

In relation to motor control, the basal ganglia have been implicated in both the scaling and focusing of movement. Hypokinetic and hyperkinetic movement disorders manifest as a consequence of overshooting and undershooting GPi (globus pallidus internus) activity thresholds, respectively. Recently, models of motor control have been borrowed to translate cognitive processes relating to the overshooting and undershooting of GPi activity, including attention and executive function. Linguistic correlates, however, are yet to be extrapolated in sufficient detail. The aims of the present investigation were to: (1) characterise cognitive-linguistic processes within hypokinetic and hyperkinetic neural systems, as defined by motor disturbances; (2) investigate the impact of surgically-induced GPi lesions upon language abilities. Two Parkinsonian cases with opposing motor symptoms (akinetic versus dystonic/dyskinetic) served as experimental subjects in this research. Assessments were conducted both prior to as well as 3 and 12 months following bilateral posteroventral pallidotomy (PVP). Reliable changes in performance (i.e. both improvements and decrements) were typically restricted to tasks demanding complex linguistic operations across subjects. Hyperkinetic motor symptoms were associated with an initial overall improvement in complex language function as a consequence of bilateral PVP, which diminished over time, suggesting a decrescendo effect relative to surgical beneficence. In contrast, hypokinetic symptoms were associated with a more stable longitudinal linguistic profile, albeit defined by higher proportions of reliable decline versus improvement in postoperative assessment scores. The above findings endorsed the integration of the GPi within cognitive mechanisms involved in the arbitration of complex language functions. In relation to models of motor control, 'focusing' was postulated to represent the neural processes underpinning lexical-semantic manipulation, and 'scaling' the potential allocation of cognitive resources during the mediation of high-level linguistic tasks. (c) 2005 Elsevier Ltd. All rights reserved.

Specialization in pyramidal cell structure in the sensory-motor cortex of the vervet monkey (Cercopethicus pygerythrus)

Recent studies have revealed systematic differences in the pyramidal cell structure between functionally related cortical areas of primates. Trends for a parallel in pyramidal cell structure and functional complexity have been reported in visual, somatosensory, motor, cingulate and prefrontal cortex in the macaque monkey cortex. These specializations in structure have been interpreted as being fundamental in determining cellular and systems function, endowing circuits in these different cortical areas with different computational power. In the present study we extend our initial finding of systematic specialization of pyramidal cell structure in sensory-motor cortex in the macaque monkey [Cereb Cortex 12 (2002) 1071] to the vervet monkey. More specifically, we investigated pyramidal cell structure in somatosensory and motor areas 1/2, 5, 7, 4 and 6. Neurones in fixed, flat-mounted, cortical slices were injected intracellularly with Lucifer Yellow and processed for a light-stable 3,3'-diaminobenzidine reaction product. The size of, number of branches in, and spine density of the basal dendritic arbors varied systematically such that there was a trend for increasing complexity in arbor structure with progression through 1/2, 5 and 7. In addition, cells in area 6 were larger, more branched, and more spinous than those in area 4. (c) 2005 IBRO. Published by Elsevier Ltd. All rights reserved.

Alcoholic neurobiology: Changes in dependence and recovery

This article presents the proceedings of a symposium held at the meeting of the International Society for Biomedical Research on Alcoholism (ISBRA) in Mannheim, Germany, in October, 2004. Chronic alcoholism follows a fluctuating course, which provides a naturalistic experiment in vulnerability, resilience, and recovery of human neural systems in response to presence, absence, and history of the neurotoxic effects of alcoholism. Alcohol dependence is a progressive chronic disease that is associated with changes in neuroanatomy, neurophysiology, neural gene expression, psychology, and behavior. Specifically, alcohol dependence is characterized by a neuropsychological profile of mild to moderate impairment in executive functions, visuospatial abilities, and postural stability, together with relative sparing of declarative memory, language skills, and primary motor and perceptual abilities. Recovery from alcoholism is associated with a partial reversal of CNS deficits that occur in alcoholism. The reversal of deficits during recovery from alcoholism indicates that brain structure is capable of repair and restructuring in response to insult in adulthood. Indirect support of this repair model derives from studies of selective neuropsychological processes, structural and functional neuroimaging studies, and preclinical studies on degeneration and regeneration during the development of alcohol dependence and recovery from dependence. Genetics and brain regional specificity contribute to unique changes in neuropsychology and neuroanatomy in alcoholism and recovery. This symposium includes state-of-the-art presentations on changes that occur during active alcoholism as well as those that may occur during recovery-abstinence from alcohol dependence. Included are human neuroimaging and neuropsychological assessments, changes in human brain gene expression, allelic combinations of genes associated with alcohol dependence and preclinical studies investigating mechanisms of alcohol induced neurotoxicity, and neuroprogenetor cell expansion during recovery from alcohol dependence.

Kinematic analysis of jaw function in children following traumatic brain injury

Primary objective: To investigate jaw movements in children following traumatic brain injury (TBI) during speech using electromagnetic articulography (EMA). Methods and procedures: Jaw movements of two non-dysarthric children ( aged 12.75 and 13.08 years) who had sustained a TBI were recorded using the AG-100 EMA system (Carstens Medizineletronik) during word-initial consonant productions. Mean quantitative kinematic parameters and coefficient of variation ( variability) values were calculated and individually compared to the mean values obtained by a group of six control children ( mean age 12.57 years, SD 1.52). Main outcomes and results: The two children with TBI exhibited word-initial consonant jaw movement durations that were comparable to the control children, with sub-clinical reductions in speed being offset by reduced distances. Differences were observed between the two children in jaw kinematic variability, with one child exhibiting increased variability, while the other child demonstrated reduced or comparable variability compared to the control group. Conclusions: Possible sub-clinical impairments of jaw movement for speech were exhibited by two children who had sustained a TBI, providing insight into the consequences of TBI on speech motor control development.

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μ M lead chloride and 0.05 μ M lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 μ M lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 μ M. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μ M and reached half-maximal inhibition of motility at about 50 μ M. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mal. Mutagen. 45:346-353, 2005. © 2005 Wiley-Liss, Inc.