[How healthy are German schools? Trends from the years 2002 to 2010]. / Wie gesund sind deutsche Schulen? Entwicklungstrends von 2002 bis 2010.

This study aims to analyse the time trends of several conditions of the school environment (satisfaction with school, school demands, quality of instruction, classmate support) in Germany that are known to affect the health of pupils.We used the national German data of the Health-Behaviour in School-aged Children (HBSC) studies conducted in the years 2002, 2006 und 2010. The time trends of these four var-iables are described by using linear and logistic regression analyses considering survey year, age group (11, 13, 15 years), gender, and school track as independent variables.We found an increase of the perceived quality of instruction and of the student's satisfaction with school from the year 2002 to the year 2010. Furthermore, pupils report slightly less support from their classmates in the present survey compared to 2002. There are no changes in the reported demands.These trend results are discussed in the light of the impact of the PISA study and the efforts to implement settings-based health promoting schools in Germany.

[Mobbing and violence at school. Trends from 2002 to 2010]. / Mobbing und Gewalt an Schulen. Entwicklungstrends von 2002 bis 2010.

The purpose of this study was to undertake an assessment and differentiated examination of the development of bullying and violence in schools between 2002 and 2010 in Germany.We examined the national German data of Health Behaviour in School-aged Children (HBSC) study in 2002, 2006 and 2010. A paper-pencil questionnaire was distributed to a representative sample (N=17 929) of 11-, 13- and 15-year-old school children. The evaluation of the data was done by descriptive statistics and logistic regression analyses, controlled by age, gender, family affluence, school type and survey year.A clear positive trend could be identified: from 2002 to 2010 the number of bullies and bully victims decreased whereas the group of the uninvolved pupils increased. There was a delay in this trend for children with low family affluence.The obvious success in the prevention of violence is shown by the decreasing rate of bullies. The paper discusses whether future prevention should focus more on victims and children with educationally deprived background.

[The HBSC Study in Germany--study design and methodology]. / Die HBSC-Studie in Deutschland--Studiendesign und Methodik.

The aim of the HBSC-Study is to collect data on the physical and mental health and health behaviour of children and adolescents and to gain a deeper insight into their situation and the specific environment they grow up in. The HBSC-study is an international school-based cross-sectional survey conducted in collaboration with the World Health Organization (WHO). The survey takes place every 4 years since 1982 and is based on a standardised protocol. In Germany the survey was first conducted in 1994 as a pilot study in North Rhine-Westphalia. The German sample is based on a random sample of classes in all public schools in Germany. 11-, 13-, and 15-year-old pupils are surveyed by means of a paper and pencil questionnaire. The questionnaire comprises a broad selection of -topics, including sociodemographics, health and risk behaviours, family, school and peers. The reported trends in the supplement are based on the data from surveys in 2002 (N=5.650), 2006 (N=7.274) and 2010 (N=5.005). The representative samples for each of the survey years are defined as follows: in 2002 the data is based on information collected in 4 Federal States (Berlin, Hesse, North Rhine-Westphalia, Saxony); in 2006 5 states define the German data file (Berlin, Hamburg, Hesse, North Rhine-Westphalia, Saxony). The data from the 2010 survey comprises data from 15 Federal States. The HBSC-data contributes towards a better understanding of the relationship between health and living conditions of young people. The papers in this supplement deliver important insights into the living context of young people and in doing this they provide important information about their health and the long-term effectiveness of public-health-measures.

[Bullying, psychosocial health and risk behaviour in adolescence]. / Bullying, psychosoziale Gesundheit und Risikoverhalten im Jugendalter.

BACKGROUND: Bullying as a subform of aggressive behaviour has not received much attention as a specific risk behaviour in adolescence. Especially the adverse health effects in relation to bullying have been barely discussed in Germany. The objective of this study is to present age- and gender-specific prevalences in bullying and to analyse the association between the different bullying roles and subjective health as well as risk behaviour. METHODS: Data were obtained from the German part of the international WHO collaborative study "Health Behaviour in School-aged Children (HBSC)" in 2002. Overall, 5,650 school children aged 11-15 years were interviewed with a standardised questionnaire. Multivariate logistic regression models were used to analyse the association between bullying, psychosocial health and risk behaviour separately for girls and boys. RESULTS: About 17% of the boys and 10% of the girls aged 11-15 years were classified as repeated bullying perpetrators. About 10% of the school children are victims of being bullied several times a month. Another 3-5% of the adolescents belonged to the group of simultaneous victims and perpetrators (bully-victims). Perpetrators as well as victims showed strong associations with psychosocial health and risk behaviour. Independently of gender, victims were significantly more likely to report repeated psychosomatic complaints, adverse mental health and negative self-reported health (boys only), than uninvolved students. Especially for male perpetrators, strong associations with regular tobacco and alcohol use and repeated drunkenness were found, while these behaviour types were significantly less prevalent among victims. The bully-victim group is characterised by high rates of psychosomatic complaints and mental health problems (boys only). CONCLUSIONS: Bullying also seems to be widespread in schools in Germany and is strongly associated with subjective health and substance-related risk behaviour. The results suggest that bullying is a critical issue that requires increasing attention in health research. The unique health problems of victims and perpetrators suggest different intervention strategies for both groups.

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis.

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Fura-2 calcium signals in skeletal muscle fibres loaded with high concentrations of EGTA.

Fura-2 is one of the most frequently used fluorescent Ca indicator dyes; yet it has limitations in tracking large intracellular Ca transients due to its high affinity for Ca. Since high affinity is of advantage when small Ca changes are to be detected, we tried the application of Fura-2 in skeletal muscle fibres which had been loaded with 15 mM internal EGTA to eliminate contractile artifacts. Under these conditions, the free Ca transients are considerably reduced in amplitude and strong saturation of Fura-2 is avoided. Cut segments of isolated muscle fibres were voltage-clamped in a double vaseline gap set-up. In the presence of high internal EGTA, free Ca (as measured with the rapid metallochromic indicator antipyrylazo III) drops rapidly from one value to a lower quasi steady-state value at the end of a depolarizing voltage pulse. This property allowed inspection of the dissociation kinetics of Ca from Fura-2 in the myoplasmic environment. The dissociation rate constant koff in the fibre was determined from the time constant of the exponential decay of the Fura-2 signal as a function of the final level of free Ca. We obtained a value of 26 s-1 at the experimental temperature of 12 degrees C. Knowledge of koff in the cell is essential for reconstructing the time course of free Ca from indicator bound Ca and for estimating the time course of the rate of release from the sarcoplasmic reticulum. The described combination of high EGTA buffering with Fura-2 fluorescence recording may be particularly useful for the determination of Ca release in small muscle cells.

Use of a metallochromic indicator to study intracellular calcium movements in skeletal muscle.

The transient increase in free myoplasmic calcium concentration due to depolarization of a skeletal muscle fibre is the net result of the release of calcium from the sarcoplasmic reticulum (SR) and its simultaneous removal by binding to various sites and by reuptake into the SR. We review here procedures recently developed in this laboratory for empirically characterizing the calcium removal processes in voltage-clamped fibres and for using such characterization to determine the time course of SR calcium release during a depolarizing pulse.

Electrical membrane properties of the muscle lamellae in Branchiostoma myotomes.

The membrane characteristics of amphioxus myotome cells were investigated using the chopped-current clamp method. The extremely thin myotome lamellae had a membrane resistance of 2.2 to 7.7 k omega cm2 and a capacity of 0.4 to 1.3 microF/micron2. The steady state current-voltage relation showed outward rectification. In Cl- - and Na+ -free solutions containing 400 mM TEA the input resistance and the time constant increased threefold and Ca2+ -dependent action potentials occurred, indicating the presence of a Ca2+ component in membrane current. It is discussed whether Ca2+ ions entering the cell during the normal action potential may significantly increase the intracellular free Ca2+ concentration.

Twitch activation in Ca2+ -free solutions in the myotomes of the lancelet (Branchiostoma lanceolatum).

The question of whether a Ca2+ influx is necessary to activate twitches in the very thin myotome cells of the lancelet was reinvestigated. Though twitches were blocked in EGTA containing Ca2+- free bathing solutions, they reappeared under certain conditions: a) when the Mg2+ level was lowered, b) when small amounts of caffeine were added, and c) when Cl- in the bathing solution was partly replaced by SCN-. In Ca2+ -containing solutions the changes a) to c) increased the twitch height. The results suggest that a Ca2+ release from the intracellular stores is involved in twitch activation and that external Ca2+ modifies the coupling between excitation and Ca2+ release rather than initiating contractile activation.

Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor alpha1 subunits (R528H)

Rabbit cDNA of the alpha1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L-type calcium currents and transients were recorded for the wild type and a mutant alpha1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L-type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L-type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura-2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L-type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T-type current and changes in the expression level of the third-type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis.

Applications of SNMS in archaeometry.

The recently developed High Frequency Mode HFM of electron gas SNMS allows investigations on insulating samples with the well known advantages of the SNMS Direct Bombardment Mode DBM for the analysis of conducting samples. HFM has been applied to analyses of different historic ceramic and glass samples in order to demonstrate the possibilities of SNMS in this field. It is shown that manufacturing places of ceramic samples could be distinguished by SNMS mass spectra ("fingerprints"). Furthermore questions of the constituents of colour remains on a painted ceramic ("Kaisermedaillon") could be answered by our SNMS analyses. SNMS investigations have been also applied to corrosion phenomena on different glass samples.

[Violence in the school--an analysis and prevention]. / Gewalt in der Schule--Analyse und Prävention.

The "Forschungsgruppe Schulevaluation" (Research Group for School Evaluation, Technical University of Dresden) conducted several empirical investigations which led to rich knowledge concerning the amount of violence in schools, the different forms of violence, roles that actors of and sufferers from violent action play and the causes for violence. Besides the wellknown socialisatory influence of families, media consumption and peers for the formation of violent behaviour of pupils and of behaviour that has an affinity to violence a causal influence of schools was detected. Questions concerning the prevention of violence, the conducting of pilot studies on the latter point in one single school as well as the evaluation of the preventory measures taken were important points in our work. In the present study we will present the most important empirical results and our experience with the prevention of violence.

A possible role of the junctional face protein JP-45 in modulating Ca2+ release in skeletal muscle.

We investigated the functional role of JP-45, a recently discovered protein of the junctional face membrane (JFM) of skeletal muscle. For this purpose, we expressed JP-45 C-terminally tagged with the fluorescent protein DsRed2 by nuclear microinjection in myotubes derived from the C2C12 skeletal muscle cell line and performed whole-cell voltage-clamp experiments. We recorded in parallel cell membrane currents and Ca(2+) signals using fura-2 during step depolarization. It was found that properties of the voltage-activated Ca(2+) current were not significantly changed in JP-45-DsRed2-expressing C2C12 myotubes whereas the amplitude of depolarization-induced Ca(2+) transient was decreased compared to control myotubes expressing only DsRed2. Converting Ca(2+) transients to Ca(2+) input flux using a model fit approach to quantify Ca(2+) removal, the change could be attributed to an alteration in voltage-activated Ca(2+) permeability rather than to altered removal properties or a lower Ca(2+) content of the sarcoplasmic reticulum (SR). Determining non-linear capacitive currents revealed a reduction of Ca(2+) permeability per voltage-sensor charge. The results may be explained by a modulatory effect of JP-45 related to its reported in vitro interaction with the dihydropyridine receptor and the SR Ca(2+) binding protein calsequestrin (CSQ).

Voltage-controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse.

The voltage-activated fluxes of Ca(2+) from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two-electrode technique. The fibres were perfused from the current-passing micropipette with a solution containing 15 mm EGTA and 0.2 mm of either fura-2 or the faster, lower affinity indicator fura-FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca(2+) inward current exhibiting half-maximal activation at -7.60 +/- 1.29 and 3.0 +/- 1.44 mV, respectively. The rate of Ca(2+) release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca(2+) removal. Results obtained with the two indicators were similar. Ca(2+) release was 2-3 orders of magnitude larger than the flux carried by the L-type Ca(2+) current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca(2+) content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation-contraction coupling in future studies of genetically altered mice.

Ca2+ current in myotome cells of the lancelet (Branchiostoma lanceolatum).

1. By using the whole-cell patch clamp method Ca2+ and Ba2+ currents were measured in the extremely thin twitch muscle cells of the protochordate Branchiostoma lanceolatum whose Ca2+ channels are likely to resemble the evolutionary ancestors of those found in vertebrate skeletal muscle. 2. When using 10 mM-Ca2+ in the artificial external solution and 1 mM-EGTA in the internal solution two kinetically different Ca2+ inward current components could be observed, showing very similar voltage dependence of activation and inactivation. 3. In solutions containing 10 mM-Ba2+ as an external charge carrier the biphasic inward current turned into a single rapidly activated and slowly inactivating current. 4. Inspecting peak currents, the voltage dependence of fractional activation and inactivation was nearly the same in Ca2+ and in Ba2+. 5. A transformation into a single component of the Ca2+ current could also be observed after perfusing the intracellular lumen with 10 mM of either EGTA or BAPTA. In the case of EGTA this transformation required considerably more time. Probably a higher internal concentration of EGTA is necessary since it binds Ca2+ more slowly than BAPTA. 6. Soon after establishing the whole-cell configuration a gradual increase of the second, slow inward current phase was observed relative to the fast component, indicating an enhancement of the slow component by intermediate intracellular buffer concentrations. 7. We conclude that the Branchiostoma myotome cells have only one Ca2+ channel system. The biphasic appearance of the inward current is caused by an unusually rapid inactivation due to Ca2+ ions, which enter the myoplasm during the current and temporarily bind to an inactivation site at the channel. The second phase probably reflects reactivation from the inactivated state upon dissociation of Ca2+ from the binding site.

Modification of excitation-contraction coupling by 4-chloro-m-cresol in voltage-clamped cut muscle fibres of the frog (R. pipiens).

1. The effect of 5 microM 4-chloro-m-cresol (4-CmC) on voltage-controlled Ca2+ release was studied in cut muscle fibres of the frog loaded with internal solutions containing 15 mM EGTA. Fibres were voltage clamped using a double Vaseline gap system, and Ca2+ signals were recorded with the fluorescent indicator dye fura-2 2. Resting intracellular free Ca2+ concentration increased from 61 to 100 nM upon application of 4-CmC. 3. Both peak rate of release of intracellularly stored Ca2+ and the steady level attained after 50 ms of depolarization increased, but the potentiation of the latter was more pronounced (by a factor of 1.7 versus 1.3). The voltage of half-maximal activation remained unchanged. 4. Non-linear intramembranous charge movements showed no significant change in voltage dependence while the maximal charge displaced by depolarization increased by 25 %. 5. The dependence of peak release flux on total intramembranous charge was not different in 4-CmC, but for the steady level of release the steepness of the relation increased by a factor of 1.3. 6. The stimulating effect of 5 microM 4-CmC on depolarization-induced Ca2+ release resembled the potentiation by 0.5 mM caffeine. However, 0.5 mM caffeine increased the peak and steady levels of the release rate by a similar factor and caused no increase in the resting free calcium concentration, indicating different modes of action of the two substances. 7. Neither 5 microM 4-CmC nor 0.5 mM caffeine led to a loss of voltage control of Ca2+ release during repolarization after short depolarizations, as has been reported previously for caffeine. Potentiated Ca2+ release could be terminated by repolarization as fast as under control conditions both with 15 mM and 0.1 mM internal EGTA. 8. The effects of 4-CmC may result from a direct opening of the release channel combined with an enhancement of the transduction mechanism that couples channel opening to displacement of voltage sensor charges.

Malignant hyperthermia and excitation-contraction coupling.

Malignant hyperthermia (MH) is a state of elevated skeletal muscle metabolism that may occur during general anaesthesia in genetically pre-disposed individuals. Malignant hyperthermia results from altered control of sarcoplasmic reticulum (SR) Ca2+ release. Mutations have been identified in MH-susceptible (MHS) individuals in two key proteins of excitation-contraction (EC) coupling, the Ca2+ release channel of the SR, ryanodine receptor type 1 (RyR1) and the alpha1-subunit of the dihydropyridine receptor (DHPR, L-type Ca2+ channel). During EC coupling, the DHPR senses the plasma membrane depolarization and transmits the information to the ryanodine receptor (RyR). As a consequence, Ca2+ is released from the terminal cisternae of the SR. One of the human MH-mutations of RyR1 (Arg614Cys) is also found at the homologous location in the RyR of swine (Arg615Cys). This animal model permits the investigation of physiological consequences of the homozygously expressed mutant release channel. Of particular interest is the question of whether voltage-controlled release of Ca2+ is altered by MH-mutations in the absence of MH-triggering substances. This question has recently been addressed in this laboratory by studying Ca2+ release under voltage clamp conditions in both isolated human skeletal muscle fibres and porcine myotubes.

L-type calcium current activation in cultured human myotubes.

The time course of activation of the skeletal muscle L-type calcium channel was studied in voltage-clamped myotubes derived from human satellite cells. The slow L-type current was isolated by inactivating faster calcium current components using appropriate prepulses or by subtracting the currents not blocked by 5 microM nifedipine. The L-type current exhibited a single exponential activation and time constants which showed little voltage dependence in the range +10 to +50mV. Currents blocked by nifedipine could be partially restored by UV-light flash photolysis. When a flash of light was applied during a depolarizing step, the activation time course of the resulting inward current contained a rapid, almost instantaneous component followed by a slower component. The amplitude of the rapid component was different when the flash was applied at different times during the depolarizing step: depolarization first increased and then decreased the fraction of channels which could rapidly be restored from the block by photolysis. Plotted versus time after the onset of the depolarization this fraction closely matched the time course of the L-type current obtained before the block by nifedipine. This indicates that the slow gating recations of the Ca2+ channel remain functional in the nifedipine-blocked state. Large conditioning depolarizations which had been shown to enhance the speed of L-type current activation in frog muscle fibres showed no effect in human myotubes. Numerical simulations using a gating scheme proposed for frog muscle demonstrate that such differences can be caused by changing just a single kinetic parameter.

Numerical methods to determine calcium release flux from calcium transients in muscle cells.

Several methods are currently in use to estimate the rate of depolarization-induced calcium release in muscle cells from measured calcium transients. One approach first characterizes calcium removal of the cell. This is done by determining parameters of a reaction scheme from a fit to the decay of elevated calcium after the depolarizing stimulus. In a second step, the release rate during depolarization is estimated based on the fitted model. Using simulated calcium transients with known underlying release rates, we tested the fidelity of this analysis in determining the time course of calcium release under different conditions. The analysis reproduced in a satisfactory way the characteristics of the input release rate, even when the assumption that release had ended before the start of the fitting interval was severely violated. Equally good reconstructions of the release rate time course could be obtained when the model used for the analysis differed in structure from the one used for simulating the data. We tested the application of a new strategy (multiple shooting) for fitting parameters in nonlinear differential equation systems. This procedure rendered the analysis less sensitive to ill-chosen initial guesses of the parameters and to noise. A locally adaptive kernel estimator for calculating numerical derivatives allowed good reconstructions of the original release rate time course from noisy calcium transients when other methods failed.