Electrostatic interactions between single arginine and phospholipids modulate physiological properties of sarcoplasmic reticulum Ca-ATPase.

Arg324 of sarcoplasmic reticulum Ca-ATPase forms electrostatic interactions with the phosphate moiety of phospholipids in most reaction states, and a hydrogen bond with Tyr122 in other states. Using site-directed mutagenesis, we explored the functional roles of Arg324 interactions, especially those with lipids, which at first glance might seem too weak to modulate the function of such a large membrane protein. The hydrogen bond forms transiently and facilitates Ca binding from the cytoplasmic side.

Angle change of the A-domain in a single SERCA1a molecule detected by defocused orientation imaging.

The sarcoendoplasmic reticulum Ca-ATPase (SERCA) transports Ca ions across the membrane coupled with ATP hydrolysis. Crystal structures of ligand-stabilized molecules indicate that the movement of actuator (A) domain plays a crucial role in Ca translocation. However, the actual structural movements during the transitions between intermediates remain uncertain, in particular, the structure of E2PCa has not been solved.

Nanodisc-based kinetic assays reveal distinct effects of phospholipid headgroups on the phosphoenzyme transition of sarcoplasmic reticulum Ca-ATPase.

Sarco(endo)plasmic reticulum Ca-ATPase catalyzes ATP-driven Ca transport from the cytoplasm to the lumen and is critical for a range of cell functions, including muscle relaxation. Here, we investigated the effects of the headgroups of the 1-palmitoyl-2-oleoyl glycerophospholipids phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylglycerol (PG) on sarcoplasmic reticulum (SR) Ca-ATPase embedded into a nanodisc, a lipid-bilayer construct harboring the specific lipid. We found that Ca-ATPase activity in a PC bilayer is comparable with that of SR vesicles and is suppressed in the other phospholipids, especially in PS.

Membrane Perturbation of ADP-insensitive Phosphoenzyme of Ca-ATPase Modifies Gathering of Transmembrane Helix M2 with Cytoplasmic Domains and Luminal Gating.

Ca transport by sarcoplasmic reticulum Ca-ATPase involves ATP-dependent phosphorylation of a catalytic aspartic acid residue. The key process, luminal Ca release occurs upon phosphoenzyme isomerization, abbreviated as E1PCa (reactive to ADP regenerating ATP and with two occluded Ca at transport sites) → E2P (insensitive to ADP and after Ca release). The isomerization involves gathering of cytoplasmic actuator and phosphorylation domains with second transmembrane helix (M2), and is epitomized by protection of a Leu-proteinase K (prtK) cleavage site on M2.

[EEG Beta and Gamma Powers When Comparing Certain Psychophysiological States with Normal and Weakened Electromyogram of Facial Muscles.].

The aim of the present study was to investigate EMG contamination on high-frequency scalp electroencephalogram (EEG) during comparisons of certain cognitive tasks performance. 19 healthy women who performed similar test tasks before and after cosmetic injections of Dysport in various face regions for reduction of facial muscles activity took part in the study. The test tasks were focused on induction of emotional states with different valences, on memory storing and extraction of verbal information.

When EMG contamination does not necessarily hide high-frequency EEG: scalp electrical recordings before and after Dysport injections.

The main aim of the present study was to investigate effects of partial reductions of electromyogram (EMG) on high-frequency scalp electroencephalogram (EEG) at rest and during performance of certain cognitive tasks. Nineteen healthy women performed the same cognitive tasks before and after cosmetic injections of Dysport in certain sites of facial muscles. Scalp EEG and EMG were recorded.

Glycine 105 as Pivot for a Critical Knee-like Joint between Cytoplasmic and Transmembrane Segments of the Second Transmembrane Helix in Ca2+-ATPase.

The cytoplasmic actuator domain of the sarco(endo)plasmic reticulum Ca-ATPase undergoes large rotational movements that influence the distant transmembrane transport sites, and a long second transmembrane helix (M2) connected with this domain plays critical roles in transmitting motions between the cytoplasmic catalytic domains and transport sites. Here we explore possible structural roles of Gly between the cytoplasmic (M2c) and transmembrane (M2m) segments of M2 by introducing mutations that limit/increase conformational freedom. Alanine substitution G105A markedly retards isomerization of the phosphoenzyme intermediate (E1PCa → E2PCa → E2P + 2Ca), and disrupts Ca occlusion in E1PCa and E2PCa at the transport sites uncoupling ATP hydrolysis and Ca transport.

The Use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases.

The membrane-bound protein family, P-type ATPases, couples ATP hydrolysis with substrate transport across the membrane and forms an obligatory auto-phosphorylated intermediate in the transport cycle. The metal fluoride compounds, BeF x , AlF x , and MgF x , as phosphate analogs stabilize different enzyme structural states in the phosphoryl transfer/hydrolysis reactions, thereby fixing otherwise short-lived intermediate and transient structural states and enabling their biochemical and atomic-level crystallographic studies. The compounds thus make an essential contribution for understanding of the ATP-driven transport mechanism.

Assembly of a Tyr122 Hydrophobic Cluster in Sarcoplasmic Reticulum Ca2+-ATPase Synchronizes Ca2+ Affinity Reduction and Release with Phosphoenzyme Isomerization.

The mechanism whereby events in and around the catalytic site/head of Ca(2+)-ATPase effect Ca(2+) release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca(2+) by taking advantage of its rapid occlusion upon formation of E1PCa2 and of stabilization afforded by a high concentration of Ca(2+). The assay is applicable to minute amounts of Ca(2+)-ATPase expressed in COS-1 cells.

[Injuries in children and adolescents in emergency services].

Background: A differentiated knowledge of trauma in children and adolescents is essential for the treatment of injured minors. The aim of this study was to present the focus of treatment in trauma emergency services.

Material And Methods: Over a period of 2 years all acutely injured children and adolescents (n = 4784) in the emergency service were analyzed prospectively.

[Event-related brain potentials when Russian verbs being conjugated: to the problem of language processing modularity].

In the light of alternative conceptions of "two-system" and "single-system" models of language processing the efforts have been undertaken to study brain mechanisnis for generation of regular and irregular forms of Russian verbs. The 19 EEG channels of evoked activity were registered along with casual alternations of speech morphology operations to be compared. Verbs of imperfective aspect in the form of an infinitive, belonging either to a group of productive verbs (default, conventionally regular class), or toan unproductive group of verbs (conventionally irregular class) were presented to healthy subjects.

[Statistically significant reproducible differences of pericranial muscles EMG in beta and gamma frequency ranges when different emotional and cognitive states are compared].

Results of comparison of power of EMG of six pericranial muscles in electroencephalographic frequency ranges beta1, beta2 and gamma are presented corresponding to performances of the tasks bound to inductions of affective experiences-external by means of presentation of images and internal by means of autobiographical memoirs revived. The tasks were focused on an induction of affective experiences with different emotional valencies--positive, negative and neutral. The EMG was derived, registered and processed by means of a computer electroencephalography techniques.

Second transmembrane helix (M2) and long range coupling in Ca²⁺-ATPase.

The actuator (A) domain of sarco(endo)plasmic reticulum Ca(2+)-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca(2+) transport despite reasonable ATPase activity, indicating that they are uncoupled.

Roles of long-range electrostatic domain interactions and K+ in phosphoenzyme transition of Ca2+-ATPase.

Sarcoplasmic reticulum Ca(2+)-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca(2+) transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.

[Induction of emotional states during oral reading of texts with different emotional valence and EEG power dynamics in frequency bands beta2 and gamma].

EEG power in frequency bands beta2 (18.5-29.5 Hz) and low gamma (30-40 Hz) was compared for situations while reading aloud with the technique "self-regulative utterance" texts as follow: a text with neutral emotional-semantic dominant; literary texts with either a positive or a negative emotional-semantic dominant; personal texts--recollections with similar dominants.

[EEG changes with unblocking of acoustic and visual sensory canals].

Modally specific and supramodal components of EEG dynamics, related to involuntary reorientation of anticipatory attention from internal into external, were studied using unblocking of either visual or acoustic apparatus. EEG registration took place while the examinees were in the resting states: with opened eyes; with closed eyes; with closed eyes and inserted noise-protective earplugs. Averaged values of EEG power in each of the derivations and of EEG coherence in each of the derivation pairs were calculated for an every examinee and for each of the states.

[Effects of unstructured video exposure on EEG power in situations of forced attention and rest].

Group 1 (N = 30) and group 2 (N = 22) of healthy volunteers participated in the experiment. EEG registration took place while the examinees were in the resting states: with closed eyes; with opened eyes; with opened eyes and being under exposure to TV channel noises (white noise). Group 1 had also to fulfill a task to count randomly appearing symbols on a screen and group 2 had to fulfill a task to find an image in the noises.

[Electroencephalographic characteristic of cognitive-specific alerting attention in verbal learning--III: Localized characteristics of EEG spatial synchronization].

Electroencephalograms (EEG) were recorder in 19 standard derivations in 88 healthy subjects, while they were in the states: rest with eyes open; memorization (learning) of verbal bilingual semantic pairs (Latin and Russian languages); the retrieval of the rote information from memory (control). We compared estimates of EEG coherence in these states for the frequency bands theta (4-7 Hz), alpha-1 (7-10 Hz), alpha-2 (10-13 Hz), beta-1 (13-18 Hz), beta-2 (18-30 Hz), gamma (30-40 Hz). When compared with the rest most strongly expressed: for memorization a decrease of coherence in the pairs of derivations from frontal and central areas of the cortex in the EEG frequency bands; for retrieval an increase of coherence in interhemispheric derivation pairs of pariental-occipital region in majority of the frequency bands.

Ca2+ release to lumen from ADP-sensitive phosphoenzyme E1PCa2 without bound K+ of sarcoplasmic reticulum Ca2+-ATPase.

During Ca(2+) transport by sarcoplasmic reticulum Ca(2+)-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca(2+) release into the lumen. Here, we find that in the absence of K(+), Ca(2+) release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca(2+) release pathway is open to some extent in the K(+)-free E1PCa(2) structure.

[EEG changes in comparison of rest states with open and closed eyes in complete darkness].

There are visible changes in EEG in transition from an awake resting state with eyes closed (REC) to an awake resting state with eyes opened (REO). These changes are usually treated as reflexion of brain activity reorganization in reply to visual perception. In present investigation we examined EEG records in awake resting states with opened and closed eyes in complete darkness.

Stable structural analog of Ca2+-ATPase ADP-insensitive phosphoenzyme with occluded Ca2+ formed by elongation of A-domain/M1'-linker and beryllium fluoride binding.

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca(2+) at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca(2+)-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca(2+) deocclusion/release to the lumen; E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeF(x)) to the phosphorylation site of the Ca(2+)-bound ATPase (E1Ca(2)).

[Mechanism of ca(2+) pump as revealed by mutations, development of stable analogs of phosphorylated intermediates, and their structural analyses].

Sarco(endo)plasmic reticulum Ca(2+)-ATPase is a representative member of P-type cation transporting ATPases and catalyzes Ca(2+) transport coupled with ATP hydrolysis. The ATPase possesses three cytoplasmic domains (N, P, and A) and ten transmembrane helices (M1-M10). Ca(2+) binding at the transport sites in the transmembrane domain activates the ATPase and then the catalytic aspartate is auto-phosphorylated to form the phosphorylated intermediate (EP).

A time-resolved spectroscopic diagnostic based on fast scintillator and optical fiber array for z-pinch plasmas.

We report a specially designed type of temporal resolved x-ray spectroscopic diagnostic using a spherically bent quartz crystal for z-pinch plasmas. Registration of time-resolved spectra was accomplished by coupling fast plastic scintillator, an optical fiber array, an optical streak camera, and a charge coupled device as the recording medium of this diagnostic. The diagnostic has been tested in imploding wire array experiments on S-300 pulsed power facility.

Roles of interaction between actuator and nucleotide binding domains of sarco(endo)plasmic reticulum Ca(2+)-ATPase as revealed by single and swap mutational analyses of serine 186 and glutamate 439.

Roles of hydrogen bonding interaction between Ser(186) of the actuator (A) domain and Glu(439) of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca(2+)-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca(2+)-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wild-type rates.