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G Hellmann

Publications and source records attributed to G Hellmann.

At least 19 recordsLinked to original sources
PubMed2000

Modulation of the calmodulin-induced inhibition of sarcoplasmic reticulum calcium release channel (ryanodine receptor) by sulfhydryl oxidation in single channel current recordings and [(3)H]ryanodine binding.

The modulation of the calmodulin-induced inhibition of the calcium release channel (ryanodine receptor) by two sulfhydryl oxidizing compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and 4, 4'-dithiodipyridine (4,4'-DTDP) was determined by single channel current recordings with the purified and reconstituted calcium release channel from rabbit skeletal muscle sarcoplasmic reticulum (HSR) and [(3)H]ryanodine binding to HSR vesicles. 0.1 microm CaM reduced the open probability (P(o)) of the calcium release channel at maximally activating calcium concentrations (50-100 microm) from 0.502 +/- 0.02 to 0.137 +/- 0.022 (n = 28), with no effect on unitary conductance. 4-CMPS (10-40 microm) and 4,4'-DTDP (0.1-0.3 mm) induced a concentration dependent increase in P(o) (> 0.9) and caused the appearance of longer open states. CaM shifted the activation of the calcium release channel by 4-CMPS or 4,4'-DTDP to higher concentrations in single channel recordings and [(3)H]ryanodine binding. 40 microm 4-CMPS induced a near maximal (P(o) > 0.9) and 0.3 mm 4,4'-DTDP a submaximal (P(o) = 0.74) channel opening in the presence of CaM, which was reversed by the specific sulfhydryl reducing agent DTT. Neither 4-CMPS nor 4,4'-DTDP affected Ca-[(125)I]calmodulin binding to HSR. 1 mm MgCl(2) reduced P(o) from 0.53 to 0.075 and 20-40 microm 4-CMPS induced a near maximal channel activation (P(o) > 0.9). These results demonstrate that the inhibitory effect of CaM or magnesium in a physiological concentration is diminished or abolished at high concentrations of 4-CMPS or 4,4'-DTDP through oxidation of activating sulfhydryls on cysteine residues of the calcium release channel.

Animals↗
PubMed1999

Activation and inhibition of purified skeletal muscle calcium release channel by NO donors in single channel current recordings.

The actions of the nitric oxide (NO) donors 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3 methyl-1-triazine (NOC-7), S-nitrosoacetylcysteine (CySNO) and S-nitrosoglutathione (GSNO) on the purified calcium release channel (ryanodine receptor) of rabbit skeletal muscle were determined by single channel current recordings. In addition, the activation of the NO donor modulated calcium release channel by the sulfhydryl oxidizing organic mercurial compound 4-(chloromercuri)phenylsulfonic acid (4-CMPS) was investigated. NOC-7 (0.1 and 0.3 mM) and CySNO (0.4 and 0.8 mM) increased the open probability (P(o)) of the calcium release channel at activating calcium concentrations (20-100 microM Ca(2+)) by 60-100%, with no effect on the current amplitude; this activation was abolished by the specific sulfhydryl reducing agent DTT. High concentrations of CySNO (1.6-2 mM) decreased P(o). Activation by GSNO (1 mM) was observed in two thirds of the experiments, but 2 mM and 4 mM GSNO markedly reduced P(o) at activating Ca(2+) (20-100 microM). In contrast to 4-CMPS, NOC-7 or GSNO had no effect at subactivating free Ca(2+) (0.6 microM). 4-CMPS further increased the open probability of NOC-7- or CySNO-stimulated channels and reversed transiently the reduced open probability of CySNO or GSNO inhibited channels at activating free Ca(2+). High concentrations of GSNO did not prevent channel activation of 4-CMPS at subactivating free Ca(2+). The NOC-7-, CySNO- or GSNO-modified channels were completely blocked by ruthenium red. It is suggested that nitrosylation/oxidation of sulfhydryls by NO donors and oxidation of sulfhydryls by 4-CMPS affect different cysteine residues essential in the gating of the calcium release channel.

Acetylcysteine↗
PubMed1999

Multifold features determine linear equation for automatic spike detection applying neural nin interictal ECoG.

OBJECTIVE: A 3-layer detection procedure was designed including preselection applying TEMPLAS software, feature extraction and artificial neural networks to determine a fast, precise and highly selective spike algorithm. METHODS: Ten intraoperative ECoG recordings of patients with temporal lobe epilepsy were computer-assisted and evaluated by 3 experts upon preselected events. For each event, 19 features were extracted, normalized and fed into a two-layer and 3-layer feedforward, back-propagate network. The weights of the 5 best individual two-layer networks of patients were averaged separately to derive a mean network, where weights were pruned, rounded off and the configuration approximated by a linear equation. RESULTS: In addition. when investigating latency histograms, a method for multi-channel artefact detection and elimination of too close intra-channel events could be found. Out of several training trails only the mean network and the linear equation were able to generalize. In comparison with the results of 19 publications, the developed solution and the estimated overall detection rates (spikes: 81%; non-spikes: 99.3%) were found to be of high quality. The processing time is short, and therefore, the method can be used to initiate other measurements. CONCLUSION: The developed solution is a fast, precise and highly selective spike detection method.

Adult↗
PubMed1998

Modification of sulfhydryls of the skeletal muscle calcium release channel by organic mercurial compounds alters Ca2+ affinity of regulatory Ca2+ sites in single channel recordings and [3H]ryanodine binding.

The actions of two organic mercurial compounds, 4-(chloromercuri)phenyl-sulfonic acid (4-CMPS) and p-chloromercuribenzoic acid (p-CMB) on the calcium release channel (ryanodine receptor) from rabbit skeletal muscle were determined by single channel recordings with the purified calcium release channel, radioligand binding to sarcoplasmic reticulum vesicles (HSR) and calcium release from HSR. p-CMB or 4-CMPS (20-100 microM) increased the mean open probability (Po) of the calcium channel at subactivating (20 nM), maximally activating (20-100 microM and inhibitory (1-4 mM) Ca2+ concentrations, with no effect on unitary conductance. This activation was partly reversed by 2 mM DTT. Both compounds affected the channels only from the cytosolic side, but not from the trans side. 100 microM 4-CMPS caused a transient increase in Po, followed by a low activity state within 1 min. At inhibitory Ca2+ concentrations Po was increased to values observed with maximally activating Ca2+ or lower, inhibitory Ca2+ concentrations. The p-CMB/4-CMPS modified channels were ryanodine sensitive and blocked by ruthenium red. [3H]Ryanodine binding was increased up to four-fold with 3-15 microM 4-CMPS/p-CMB (Hill coefficient 1.7-2.0) at 4 microM Ca2+ and reduced at high concentrations (50-200 microM). The increase in [3H]ryanodine binding by 10 microM 4-CMPS was completely inhibited by 2 mM DTT. 4-CMPS significantly increased the affinity for the high affinity calcium activation sites and decreased the affinity of low affinity calcium inhibitory sites of specific [3H]ryanodine binding. 4-CMPS increased the affinity of the ryanodine receptor for high affinity ryanodine binding without a change in receptor density. 4-CMPS induced a rapid, concentration-dependent, biphasic calcium release from passively calcium-loaded HSR vesicles at subactivating Ca2+ concentrations (20 nM), which was partly inhibited by 4 mM DTT and completely blocked by 20 microM ruthenium red. It is suggested that the 4-CMPS-induced modulation of essential sulfhydryls involved in the gating of the calcium release channel results in a modulation of the apparent calcium affinity of the activating high affinity and inhibitory low affinity calcium binding sites of the calcium release channel.

4-Chloromercuribenzenesulfonate↗
PubMed1996

Extensible biosignal (EBS) file format: simple method for EEG data exchange.

Increasing use of computer technology in EEG research requires the creation of standardized data formats to transmit, exchange, analyze or modify mainly EEG/MEG as well as mere general polygraphic data. The extensible biosignal file format (EBS) has been designed for easy use. The concept of the EBS format is a simple structure of variable size, consisting of one fixed and two variable headers and a data section. In the variable header, any information can be stored in attributes. The data are archived in one of 3 organizational forms: channel order, temporal order, or compressed. The format supports various data types, multiple biosignals (ECG, EEG, MEG, polygraph), annotations, processing history, location diagrams (CGM), 16 hit ISO 10646 character set, random access to large amounts of data, global or private extensions, self-identification, and multiple tools for conversion, modification and visualization which are freely available in source code.

Brain↗
PubMed1993

Phosphorylation of serine 2843 in ryanodine receptor-calcium release channel of skeletal muscle by cAMP-, cGMP- and CaM-dependent protein kinase.

The aim of the present study was to determine the phosphorylation of the purified ryanodine receptor-calcium release channel (RyR) of rabbit skeletal muscle sarcoplasmic reticulum by the cAMP-dependent protein kinase (PK-A), cGMP-dependent protein kinase (PK-G) and Ca(2+)-, CaM-dependent protein kinase (PK-CaM) and the localization of phosphorylation sites. Phosphorylation was highest with PK-A (about 0.9 mol phosphate/mol receptor subunit), between one-half to two-thirds with PK-G and between one-third and more than two-thirds with PK-CaM. Phosphoamino acid analysis revealed solely labeled phosphoserine with PK-A and PK-G and phosphoserine and phosphothreonine with PK-CaM. Reverse-phase high-performance liquid chromatography (HPLC) of cyanogen bromide/trypsin digests of the phosphorylated RyR (purified by gel permeation HPLC) and two-dimensional peptide maps revealed one major phosphopeptide by PK-A and PK-G phosphorylation and several labeled peaks by PK-CaM phosphorylation. Automated Edman sequence analysis of the major phosphopeptide obtained from PK-A and PK-G phosphorylation and one phosphopeptide obtained from PK-CaM phosphorylation yielded the sequence KISQTAQTYDPR (residues 2841-2852) with serine 2843 as phosphorylation site (corresponding to the consensus sequence RKIS), demonstrating that all three protein kinases phosphorylate the same serine residue in the center of the receptor subunit, a region proposed to contain the modulator binding sites of the calcium release channel.

Amino Acid Sequence↗
PubMed1990

Activation and inhibition of the calcium-release channel of isolated skeletal muscle heavy sarcoplasmic reticulum. Models of the calcium-release channel.

Calcium-independent calcium efflux from heavy sarcoplasmic reticulum (HSR) of skeletal muscle was found to be biphasic, with half-times of 2-6 s and 200-400 s for the first and second phase, respectively. Calcium-, AMP- and caffeine-induced calcium efflux was triphasic, with half-times of 0.05-0.2 s, 1-5 s and 100-400 s for the first, second and third phases, respectively. This very fast first phase is certainly due to calcium efflux via the calcium-release channel of HSR vesicles. Both ruthenium red and neomycin inhibited the first phase of the calcium-independent calcium efflux and the first phase of the calcium-, AMP- or caffeine-induced calcium efflux completely, whilst the second phase was fully inhibited by ruthenium red only and partially inhibited by neomycin at high concentrations, indicating that the second phase of calcium release also occurs via the calcium-release channel. Various models for calcium efflux through the release channel have been tested by simulation. Activation and inhibition of the channel-mediated calcium efflux from HSR cannot be explained by two states of the calcium-release channel (open or closed), but requires the existence of at least three states. A channel with one open state and two closed states, resulting in a rapid inactivation, is the most simple model compatible with the experimental data. According to this model, activation is assumed to reduce inactivation of the channel, whilst inhibition assumes an acceleration of channel inactivation. This mechanism most likely applies to neomycin. An additional open-blocked state has to be assumed for inhibition by ruthenium red.

Adenosine Monophosphate↗
PubMed1990

[Calcium channel mediated calcium release from the rabbit skeletal muscle sarcoplasmic reticulum vesicle].

Activation and inhibition of the calcium release channel of rabbit skeletal muscle heavy sarcoplasmic reticulum (HSR) was investigated by various methods. The calcium release channel is activated by binding of calcium in the micromolar range and by binding of adenine nucleotides in the millimolar range. Ruthenium red and neomycin are potent inhibitors of the channel at nanomolar to micromolar concentrations. Dantrolene inhibits the rate of caffeine-induced calcium release. Several models of the calcium release channel were considered to explain the three-phasic calcium release from HSR vesicles. Simulation of calcium efflux data according to various models suggest that the calcium release channel has at least three states. The experimental results can be explained by assuming one open and two closed states of the calcium release channel, but not by assuming one open and one closed state.

Adenine Nucleotides↗
PubMed1990

[Use of computer technique in occupational health inspection].

An organisation concept of an inspectorate of occupational hygiene is described. Its development was started any years ago, but it is reached a temporary optimum just by the introduction of computers. The present state is described in characteristics, also is concise indicated the possible and desirable further development. Interested persons may demand comprehensive informations about the total concept and documentations about the individual part-programs from the authors under send in of a diskette.

Documentation↗
PubMed1988

Inhibition of calcium release from skeletal muscle sarcoplasmic reticulum by calmodulin.

The effect of calmodulin on calcium release from heavy sarcoplasmic reticulum isolated from rabbit skeletal muscle was investigated with actively and passively calcium loaded sarcoplasmic reticulum vesicles and measured either spectrophotometrically with arsenazo III or by Millipore filtration technique. The transient calcium-, caffeine- and AMP-induced calcium release from actively loaded sarcoplasmic reticulum vesicles was reduced to 29%, 51% and 59% of the respective control value by 1 microM exogenous calmodulin. Stopped-flow measurements demonstrate that calmodulin reduces the apparent rate of caffeine-induced calcium release from actively loaded sarcoplasmic reticulum. The rate of calcium uptake measured in the presence of ruthenium red, which blocks the calcium release channel, was not affected by calmodulin or calmodulin-dependent phosphorylation of sarcoplasmic reticulum vesicles with ATP[S]. The rate of the calcium-, caffeine- and AMP-induced calcium release from passively loaded sarcoplasmic reticulum vesicles was reduced 1.4-2.0-fold by 1 microM exogenous calmodulin, i.e. the half-time of release was maximally increased by a factor of two, whilst calmodulin-dependent phosphorylation of a 57 kDa protein with ATP[S] had no effect. The data indicate that calmodulin itself regulates the calcium release channel of sarcoplasmic reticulum.

Adenosine Monophosphate↗
PubMed1986

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminoquinolines↗
PubMed1984

Calmodulin X (Ca2+)4 is the active calmodulin-calcium species activating the calcium-, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum in the regulation of the calcium pump.

Calcium-, calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum increases the rate of calcium transport. The complex dependence of calmodulin-dependent phosphoester formation on free calcium and total calmodulin concentrations can be satisfactorily explained by assuming that CaM X (Ca2+)4 is the sole calmodulin-calcium species which activates the calcium-, calmodulin-dependent, membrane-bound protein kinase. The apparent dissociation constant of the E X CaM X (Ca2+)4 complex determined from the calcium dependence of calmodulin-dependent phosphoester formation over a 100-fold range of total calmodulin concentrations (0.01-1 microM) was 0.9 nM; the respective apparent dissociation constant at 0.8 mM free calcium, 1 mM free magnesium with low calmodulin concentrations (0.1-50 nM) was 2.60 nM. These results are in good agreement with the apparent dissociation constant of 2.54 nM of high affinity calmodulin binding determined by 125I-labelled calmodulin binding to sarcoplasmic reticulum fractions at 1 mM free calcium, 1 mM free magnesium and total calmodulin concentration ranging from 0.1 to 150 nM, i.e. conditions where approximately 98% of the total calmodulin is present as CaM X (Ca2+)4. The apparent dissociation constant of the calcium-free calmodulin-enzyme complex (E X CaM) is at least 100-fold greater than the apparent dissociation constant of the E X CaM X (Ca2+)4 complex, as judged from non-saturation 125I-labelled calmodulin binding at total calmodulin concentrations of up to 150 nM, in the absence of calcium.

Animals↗
PubMed1983

Correlation between calmodulin-dependent increase in the rate of calcium transport and calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum. Characterization of calmodulin-dependent phosphorylation.

The aim of the present study was to prove a correlation between the calmodulin-dependent increase in the rate of calcium transport by dog cardiac sarcoplasmic reticulum and calmodulin-dependent phosphorylation. The dependence of phosphorylation on the total calmodulin concentration at 75 microM and 1 microM free calcium gave apparent calmodulin half-saturation constants Km (CaM) of 9.4 nM and 181 nM, respectively, whilst the apparent Km (CaM) for the rate of calmodulin-stimulated calcium transport carried out at 1 microM calcium, but phosphorylated prior to the calcium uptake at 75 microM or 1 microM calcium, were 12.5 nM and 127 nM, respectively. A positive correlation was obtained between calmodulin-dependent increase in the rate of calcium transport and hydroxylamine-insensitive phosphoester formed by the calcium/calmodulin-regulated, membrane-bound protein kinase. More than 90% of incorporated [32P]phosphate is confined to a 26-28-kDa or 9-11-kDa protein as determined by polyacrylamide gel electrophoresis following solubilization in sodium dodecyl sulfate at 37 degrees C and at 100 degrees C, respectively, similar to the results obtained by phosphorylation with cAMP-dependent protein kinase. The data indicate that calmodulin-dependent phosphorylation of the above protein(s) is causally related to the stimulation of the rate of calcium transport by cardiac sarcoplasmic reticulum, which is at least partially due to a shift in the calcium dependence of the rate of calcium transport to lower free calcium concentrations, K(Ca), of 1.25 microM and 0.61 microM in controls and calmodulin-dependent phosphorylation, respectively. Activation of calmodulin-dependent phosphorylation by free calcium at total calmodulin concentrations of 300 nM, 100 nM and 30 nM gave apparent K(Ca) values of 0.83 microM, 1.44 microM and 2.3 microM and Hill coefficients of 4.13, 3.76 and 3.79, respectively, indicating that all four calcium binding sites of calmodulin have to be saturated to obtain activation of the calcium/calmodulin-regulated protein kinase. The calmodulin-dependent modulation of calcium transport in vivo is, therefore, determined to great extent by the total calmodulin concentration present in the sarcoplasm.

Animals↗