Influence of design of dentist's chairs on body posture for dentists with different working experience.

BACKGROUND: Musculoskeletal disorders (MSD) are a common health problem among dentists. Dental treatment is mainly performed in a sitting position. The aim of the study was to quantify the effect of different ergonomic chairs on the sitting position. In addition, it was tested if the sitting position of experienced workers is different from a non-dental group. METHODS: A total of 59 (28 m/31f) subjects, divided into two dentist groups according to their work experience (students and dentists (9 m/11f) < 10 years, dentists (9 m/10f) ≥ 10 years) and a control group (10 m/10f) were measured. A three-dimensional back scanner captured the bare back of all subjects sitting on six dentist's chairs of different design. Initially, inter-group comparisons per chair, firstly in the habitual and secondly in the working postures, were carried out. Furthermore, inter-chair comparison was conducted for the habitual as well as for the working postures of all subjects and for each group. Finally, a comparison between the habitual sitting posture and the working posture for each respective chair (intra-chair comparison) was conducted (for all subjects and for each group). In addition, a subjective assessment of each chair was made. For the statistical analysis, non-parametric tests were conducted and the level of significance was set at 5%. RESULTS: When comparing the three subject groups, all chairs caused a more pronounced spinal kyphosis in experienced dentists. In both conditions (habitual and working postures), a symmetrical sitting position was assumed on each chair. The inter-chair comparisons showed no differences regarding the ergonomic design of the chairs. The significances found in the inter-chair comparisons were all within the measurementerror and could, therefore, be classified as clinically irrelevant. The intra-chair comparison (habitual sitting position vs. working sitting position) illustrated position-related changes in the sagittal, but not in the transverse, plane. These changes were only position-related (forward leaned working posture) and were not influenced by the ergonomic sitting design of the respective chair. There are no differences between the groups in the subjective assessment of each chair. CONCLUSIONS: Regardless of the group or the dental experience, the ergonomic design of the dentist's chair had only a marginal influence on the upper body posture in both the habitual and working sitting postures. Consequently, the focus of the dentist's chair, in order to minimize MSD, should concentrate on adopting a symmetrical sitting posture rather than on its ergonomic design.

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals.

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1-43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.

Differential regulation of granule-to-granule and granule-to-plasma membrane fusion during secretion from rat pituitary lactotrophs.

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, approximately 11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.

Acetylcholine hot spots: development on myotubes cultured from aneural limb buds.

The hypothesis that neural induction plays a role in the development of acetylcholine hot spots (high-sensitivity regions) was tested by electrophysiological mapping of the distribution of acetylcholine sensitivity of myotubes derived from aneural hindlimb buds of chick embryos. Hot spots were found. Therefore, hot spot development is not dependent on prior contact with nerve processes.

Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction.

The fluorescent dyes FM1-43 and RH414 label motor nerve terminals in an activity-dependent fashion that involves dye uptake by synaptic vesicles that are recycling. This allows optical monitoring of vesicle recycling in living nerve terminals to determine how recycled vesicles reenter the vesicle pool. The results suggest that recycled vesicles mix with the pool morphologically and functionally. One complete cycle of release of transmitter, recycling of a vesicle, and rerelease of transmitter appears to take about 1 minute.

How reliable are the risk estimates for X-ray examinations in forensic age estimations? A safety update.

Possible biological side effects of exposure to X-rays are stochastic effects such as carcinogenesis and genetic alterations. In recent years, a number of new studies have been published about the special cancer risk that children may suffer from diagnostic X-rays. Children and adolescents who constitute many of the probands in forensic age-estimation proceedings are considerably more sensitive to the carcinogenic risks of ionizing radiation than adults. Established doses for X-ray examinations in forensic age estimations vary from less than 0.1 microSv (left hand X-ray) up to more than 800 microSv (computed tomography). Computed tomography in children, as a relatively high-dose procedure, is of particular interest because the doses involved are near to the lower limit of the doses observed and analyzed in A-bombing survivor studies. From these studies, direct epidemiological data exist concerning the lifetime cancer risk. Since there is no medical indication for forensic age examinations, it should be stressed that only safe methods are generally acceptable. This paper reviews current knowledge on cancer risks associated with diagnostic radiation and aims to help forensic experts, dentists, and pediatricians evaluate the risk from radiation when using X-rays in age-estimation procedures.

Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction.

We used FM1-43 imaging and intracellular recordings of synaptic potentials to measure the time course of endocytosis in frog motor nerve terminals following tetanic nerve stimulation, and we used fura-2 imaging of intraterminal Ca2+ concentration to compare endocytic rate and [Ca2+]i. Following a 30 Hz tetanus, endocytosis declined exponentially with a time constant that depended on the duration of stimulation. The level of [Ca2+]i rose from a resting value of about 100 nM to more than 500 nM during 30 Hz stimulation, and rapidly declined to 200-250 nM after stimulation. [Ca2+]i returned to resting level with a time course that, like endocytosis, depended on the duration of tetanic stimulation. However, the rate of [Ca2+]i recovery was much slower than the rate of endocytosis, leading to the conclusion that endocytic rate is not determined solely by the instantaneous level of [Ca2+]i.

Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals.

We have identified and characterized two vesicle recycling pathways in frog motor nerve terminals. We exploited the differential staining properties of FM dyes of varying hydrophobicity to label selectively two different vesicle pools, using optical imaging and electron microscopy of photoconverted dyes. During a 1 min tetanus, a rapidly recycling route places vesicles selectively into a small readily releasable pool comprising about 20% of vesicles. After the tetanus, a much slower pathway (from which FM2-10 but not FM1-43 can be rinsed) delivers vesicles via infoldings and cisternae selectively to a reserve pool with a halftime of about 8 min. Mixing between the two pools is slow. During stimulation at 30 Hz, 10-15 s is required to mobilize and release dye from the reserve pool.

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.

Regulation of dense core release from neuroendocrine cells revealed by imaging single exocytic events.

Using FM1-43 fluorescence, we have optically detected single exocytic and endocytic events in rat pituitary lactotrophs. About fifty discrete fluorescent spots abruptly appear around the entire surface of a cell bathed in FM1-43 and high-potassium saline. The spots, which also immunostain for prolactin, reflect the labeling of dense cores as well as membranes of exocytosed secretory granules. Stained cores are not released, but remain attached to the cell and are eventually endocytosed. However, in cells exposed to dopamine (or an analog, bromocriptine), the cores dissolve and are secreted after several seconds. Solubilization of dense cores is mediated through a reduction in cytoplasmic cyclic AMP. Thus, the composition of secretions from individual secretory granules is regulated.

The use of traditional and complementary medicine among patients with multiple sclerosis in Belgium.

Introduction. Conventional treatment of multiple sclerosis (MS) is often disappointing. As a result, some of these patients seek salvation in traditional and complementary medicine (T&CM). The aim of this study is to describe how many patients with MS use T&CM and what their motives and expectations are in doing so. Methods. Ninety-nine patients with diagnosed MS, attending the service of ambulatory revalidation of the National Clinic for Multiple Sclerosis in Melsbroek (Belgium) were included in February 2004 in this retrospective study. All patients had MS resulting in motoric or psychosocial symptoms. The disability was not quantified for this study. Participants were interviewed by means of a structured questionnaire on their current treatment of MS including T&CM. Results. In total 44% of the participants had experiences with T&CM. The most frequently used T&CM were homeopathy and acupuncture. Participants using conventional treatment were more satisfied with the support (p=0.006) and the treatment outcome (0.018) than T&CM users. The use of T&CM was not related to gender, education, living conditions, causal treatment such as disease modifying-therapy (DMT), grade of disability or subtype of the disease. Conclusion. Patients diagnosed with MS seek hope in T&CM such as homeopathy or acupuncture. The results of this study suggest that MS patients need more professional support in their personal search for alternative therapies. Key point. 50% of patients diagnosed with multiple sclerosis search relief in traditional and complementary medicine such as homeopathy or acupuncture. These patients often feel compelled to try every opportunity to heal, often stimulated or urged on by friends or relatives. Multiple sclerosis patients are more satisfied with their conventional treatment than with the traditional and complementary medicine.

Synaptic transmission. Kinetics of synaptic-vesicle recycling.

The kinetics of different steps in synaptic-vesicle recycling, including exocytosis, internalization and repriming, have recently been estimated in various types of living cell.

Monitoring secretion in real time: capacitance, amperometry and fluorescence compared.

Techniques for measuring exocytosis, endocytosis and vesicle cycling in living cells in real time have resulted in a rapid expansion in the knowledge of these processes in neurons and other secretory cells. Several experimental approaches, developed during the past decade, have played key roles in this expansion. In this review we focus on three techniques: electrophysiological methods for monitoring membrane capacitance, electrochemical methods for detecting released secretory contents and optical methods for imaging membranes of endosomes and recycled vesicles that are stained with fluorescent dyes. Each technique has contributed unique and complementary information about the vesicle cycle, advancing our knowledge of the kinetics of membrane fusion and retrieval, the identity of the secretory contents and the spatial patterns and directional pathways involved in secretory membrane recycling. Naturally, each technique has inherent limitations; some of these shortcomings have recently been resolved by using more than one method simultaneously.

Imaging exocytosis and endocytosis.

From the secretion of neurotransmitters via synaptic vesicles to the expulsion of cellular waste via contractile vacuoles, exocytosis and its sequel, endocytosis, are being explored with a variety of new optical tools. Fluorescent markers, especially styryl dyes such as FM1-43 (which reversibly labels endosomal membranes), have been used to follow exo- and endocytic events in many cell types. Even though the development of new dyes is still largely empirical, some theoretical principles have emerged to guide future dye chemistry. Moreover, advances in optical imaging technology that augment conventional fluorescence microscopy are appearing. For example, interference reflection microscopy (which requires no flurophore) and total internal reflection microscopy have recently been used to observe single exocytic events at the contact point between a glass coverslip and the plasma membrane.

Effects of staurosporine on exocytosis and endocytosis at frog motor nerve terminals.

Observations of the dynamic staining and destaining of FM1-43 in frog motor nerve terminals (Henkel and Betz, 1995) suggested that staurosporine might shorten the interval between exocytosis and endocytosis, inducing a "kiss and run" mode of exocytosis and endocytosis. We tested this hypothesis by using FM1-43 imaging (to measure the time course of FM1-43 endocytosis), intracellular recording of evoked synaptic potentials (to measure acetylcholine release), and electron microscopy (to examine synaptic vesicle distribution). Staurosporine reduced FM1-43 uptake during but not after a tetanus, increased the speed of end plate potential (EPP) amplitude rundown, and greatly slowed the recovery from synaptic depression. Ultrastructural observations showed pronounced vesicle depletion near active zones after tetanic stimulation in staurosporine-treated preparations. These results suggest that staurosporine acted primarily to impair mobilization of synaptic vesicles during tetanic stimulation.

Evidence for active chloride accumulation in normal and denervated rat lumbrical muscle.

Intracellular Cl- activity (aiCl) was measured with Cl(-)-sensitive microelectrodes in normal and denervated rat lumbrical muscle. In normal muscle bathed in normal Krebs solution, aiCl lay close to that predicted by the Nernst equation. The addition of 9-anthracene carboxylic acid, which blocks Cl- conductance, caused aiCl to increase far above that predicted by a passive distribution. Furosemide (10 microM) reversibly blocked this accumulation. After muscle denervation, aiCl progressively increased for 1-2 wk. The rise occurred in two stages. The initial stage (1-3 d after denervation) reflected passive Cl- accumulation owing to membrane depolarization. At later times, aiCl continued to increase, with no further change in membrane potential, which suggests an active uptake mechanism. This rise approximately coincided with the natural reduction in membrane conductance to Cl- that occurs several days after denervation. Na+ replacement, K+ replacement, and furosemide each reversibly blocked the active Cl- accumulation in denervated muscle. Quantitative estimates suggested that there was little difference between Cl- flux rates in normal and denervated muscles. The results can be explained by assuming that, in normal muscle, an active accumulation mechanism operates, but that Cl- lies close to equilibrium owing to the high membrane conductance to Cl-. The rise in aiCl after denervation can be accounted for by the membrane depolarization, the reduction in membrane Cl- conductance, and the nearly unaltered action of an inwardly directed Cl- "pump."

Mapping electric currents around skeletal muscle with a vibrating probe.

A vibrating microelectrode, or vibrating probe (Jaffe and Nuccitelli, 1974), was used to map the pattern of artificially created electric currents flowing around single muscle fibers at the edge of frog cutaneous pectoris muscles. When a muscle fiber was impaled with a micropipette, a "point sink" of current was often created at the site of impalement because of injury to the cell membrane. Current, being drawn from the flanking membrane, flowed into the cell only at this point. This defined current allowed us to map the spatial resolving power of the vibrating probe by moving to different positions near the impalement site. The results suggest that under our experimental conditions the limit of resolution is a few tens of micrometers. The results were fit reasonably well by a computer model. Current was also passed through a micropipette and mapped at various positions with the vibrating probe. In this case, the current flowed to a remote reference electrode. With the current electrode in the extracellular fluid, the probe signal decayed as the inverse square of the distance, as expected. With the current electrode placed intracellularly, current was funneled along the muscle fiber axis, reflecting its cable-like properties. The signal recorded by the vibrating probe was altered accordingly, and the results could be well fit by a simple model.

Properties of an endogenous steady current in rat muscle.

A vibrating probe was used to study a steady electric current generated by isolated, whole lumbrical muscles of the rat. Spatial mapping showed that current leaves the muscle in the synaptic region and re-enters in the flanking extrajunctional regions. The point of maximum outward current coincided precisely with the endplate region. As the probe was moved radially away from the endplate region, the current declined monotonically, and the results could be fit with a simple model. As the probe was moved axially away from the endplate region, the current declined and became inward over a distance of approximately 0.5 mm. The physiological mechanism by which the current is generated was also studied. alpha-Bungarotoxin and tetrodotoxin had no significant effect on the current, which suggests that acetylcholine channels and gated sodium channels are not involved in the generation of the current. Ouabain produced a slowly developing, partial inhibition of the current, reducing it by approximately 40% over a period of 30-40 min. Carbachol produced a large inward current at the endplate region. After the carbachol action was terminated with alpha-bungarotoxin, an outward current reappeared, and a transient "overshoot" developed. During the overshoot, which lasted approximately 30-40 min, the outward current was approximately doubled. This overshoot was completely abolished by ouabain. The overshoot is interpreted as reflecting the increased activity of electrogenic sodium pumping in the endplate region, caused by the influx of Na ions during carbachol application. Because of the very different actions of ouabain on the normal current and on the overshoot after carbachol application, we concluded that the normal outward current is not produced by electrogenic sodium pumping in the endplate region.

Physiological basis of a steady endogenous current in rat lumbrical muscle.

In an attempt to determine the mechanism by which rat skeletal muscle endplates generate a steady outward current, we measured the effects of several drugs (furosemide, bumetanide, 9-anthracene carboxylic acid [9-AC]) and changes in external ion concentration (Na+, K+, Cl-, Ba++) on resting membrane potential (Vm) and on the steady outward current. Each of the following treatments caused a 10-15-mV hyperpolarization of the membrane: replacement of extracellular Cl- with isethionate, addition of furosemide or bumetanide, and addition of 9-AC. These results suggest that Cl- is actively accumulated by the muscle fibers and that the equilibrium potential of Cl- is more positive than the membrane potential. Removal of external Na+ also caused a large hyperpolarization and is consistent with evidence in other tissues that active Cl- accumulation requires external Na+. The same treatments greatly reduced or abolished the steady outward current, with a time course that paralleled the changes in Vm. These results cannot be explained by a model in which the steady outward current is assumed to arise as a result of a nonuniform distribution of Na+ conductance, but they are consistent with models in which the steady current is produced by a nonuniform distribution of GCl or GK. Other treatments (Na+-free and K+-free solutions, and 50 microM BaCl2) caused a temporary reversal of the steady current. Parallel measurements of Vm suggested that in none of these cases did the electrochemical driving force for K+ change sign, which makes it unlikely that the steady current arises as a result of a nonuniform distribution of GK. All of the results, however, are consistent with a model in which the steady outward current arises as a result of a nonuniform distribution of Cl- conductance, with GCl lower near the endplate than in extrajunctional regions.

Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43.

The neurotoxin Black Widow Spider Venom (BWSV) triggers massive release of neurotransmitter at synapses. Here we demonstrate that the action of BWSV on the frog neuromuscular junction can be visualized in vivo by the use of the fluorescent styryl dye FM1-43. This vital dye stains recycled synaptic vesicles upon nerve stimulation. Motor nerve terminals were stained with FM1-43 via electrical stimulation, washed and then exposed to BWSV or alpha-Latrotoxin. All terminals destained completely, independent of external calcium. Exposure of frog nerve terminals to BWSV in the presence of FM1-43 and calcium led to staining of terminals. The staining pattern appeared to be exactly the same as in control preparations, stimulated electrically via the nerve. When the same experiment was performed in the absence of calcium, only a minute quantity of dye was taken up into the nerve terminals, and the synapses looked swollen and puffed. Addition of external calcium to these preparations elicited an immediate shrinking of the nerve terminals, indicating endocytosis. These observations support electron-microscopic data that suggest an important role for extracellular calcium in endocytosis of BWSV poisoned nerve terminals.