Electrical Grounding Improves Vagal Tone in Preterm Infants.

Background: Low vagal tone (VT) is a marker of vulnerability to stress and the risk of developing necrotizing enterocolitis in preterm infants. Electric fields produced by equipment in the neonatal intensive care unit (NICU) induce an electric potential measurable on the skin in reference to ground. An electrical connection to ground reduces the skin potential and improves VT in adults.

Cooperative effects of tropomyosin on the dynamics of the actin filament.

Tropomyosin (Tpm) plays an important role in regulating the organisation and functions of the actin cytoskeleton. Here, we describe a new approach to analyse the effects of Tpm on actin dynamics. Using F-actin proteolytically modified within the DNase-binding loop (ECP-actin), we show that Tpm binding almost completely suppresses the increased subunit exchange intrinsic for this F-actin.

Four paralog gelsolin genes are differentially expressed in the earthworm Lumbricus terrestris.

We have identified and characterized four distinct variants of the gelsolin-related protein (EWAM P1-P4) in the earthworm L. terrestris. All of these proteins biochemically qualify as gelsolins since they sever actin filaments in a calcium dependent manner.

The interaction of gelsolin with tropomyosin modulates actin dynamics.

We have investigated the interactions between the actin-binding proteins gelsolin and tropomyosin, with special respect to any effects on the functional properties of gelsolin. Limited proteolysis indicated that the loop connecting the gelsolin domains G3 and G4 is involved in tropomyosin binding. Under nonpolymerizing conditions, binding of tropomyosin neither prevented the formation of a 2: 1actin-gelsolin complex, nor did it affect the nucleating activity of gelsolin in actin polymerization, likely as a result of competitive displacement of tropomyosin from gelsolin.

Isoforms of gelsolin from lobster striated muscles differ in calcium-dependence.

Two distinct isoforms of the Ca-dependent actin filament severing protein gelsolin were identified in cross-striated muscles of the American lobster. The variants (termed LG1 and LG2) differ by an extension of 18 AA at the C-terminus of LG1, and by two substitutions at AA735 and AA736, the two C-terminal amino acids of LG2. Functional comparison of the isolated and purified proteins revealed gelsolin-typical properties for both with differences in Ca(2+)-sensitivity, LG2 being activated at significant lower Ca-concentration than LG1: Half maximal activation for both filament severing and G-actin binding was ∼4×10(-7)M Ca(2+) for LG2 vs.

Electron-microscopical localization of gelsolin in various crustacean muscles.

Gelsolin was localized by immunoelectron microscopy in fast and slow cross-striated muscles of the lobster Homarus americanus. When ultrathin sections of the muscles were labelled with anti-gelsolin and a gold-conjugated second antibody, 90% of all gold particles in the myoplasm were detected on myofibrils, preferentially in the I-band and AI-region of the sarcomeres. Both the region of the H-zone (lacking thin filaments) and the Z-disc contained no or little gold label.

Difference in polymerization and steady-state dynamics of free and gelsolin-capped filaments formed by alpha- and beta-isoactins.

The polymerization of scallop beta-like actin is significantly slower than that of skeletal muscle alpha-actin. To reveal which steps of polymerization contribute to this difference, we estimated the efficiency of nucleation of the two actins, the rates of filament elongation at spontaneous and gelsolin-nucleated polymerization and the turnover rates of the filament subunits at steady-state. Scallop actin nucleated nearly twice less efficient than rabbit actin.

Involvement of a gelsolin-related protein in spermatogenesis of the earthworm Lumbricus terrestris.

A gelsolin-related protein was isolated from seminal vesicles of the annelid Lumbricus terrestris. Compared with the isoforms of the gelsolin-related protein previously found in the muscle of the annelid body wall, the isolated protein was assigned to the first isoform (EWAM-P1) because of its electrophoretic mobility, chromatographic elution behaviour, immunological cross-reactivity and identical nucleotide sequence of segments obtained by reverse transcription/polymerase chain reaction. Immunofluorescence studies with smear preparations of developing male germ cells revealed characteristic changes of the local distribution of actin and EWAM-P1 during spermatogenesis.

Calcium-induced conformational changes in the C-terminal half of gelsolin stabilize its interaction with the actin monomer.

The basic mechanism for the nucleating effect of gelsolin on actin polymerization is the formation of a complex of gelsolin with two actin monomers. Probably due to changes in the C-terminal part of gelsolin, a stable ternary complex is only formed at [Ca(2+)] >10(-5) M [Khaitlina, S., and Hinssen, H.

Topological assignment of the N-terminal extension of plasma gelsolin to the gelsolin surface.

The actin-binding protein gelsolin is highly conserved in vertebrates and exists in two isoforms, a cytoplasmic and an extracellular variant, generated by alternative splicing. In mammals, these isoforms differ only by an N-terminal extension in plasma gelsolin, a short sequence of up to 25 amino acids. Cells and tissues may contain both variants, as plasma gelsolin is secreted by many cell types.

Damped elastic recoil of the titin spring in myofibrils of human myocardium.

The giant protein titin functions as a molecular spring in muscle and is responsible for most of the passive tension of myocardium. Because the titin spring is extended during diastolic stretch, it will recoil elastically during systole and potentially may influence the overall shortening behavior of cardiac muscle. Here, titin elastic recoil was quantified in single human heart myofibrils by using a high-speed charge-coupled device-line camera and a nanonewtonrange force sensor.

Regulation of sodium channel activity by capping of actin filaments.

Ion transport in various tissues can be regulated by the cortical actin cytoskeleton. Specifically, involvement of actin dynamics in the regulation of nonvoltage-gated sodium channels has been shown. Herein, inside-out patch clamp experiments were performed to study the effect of the heterodimeric actin capping protein CapZ on sodium channel regulation in leukemia K562 cells.

Nebulin is a thin filament protein of the cardiac muscle of the agnathans.

Nebulin is an integral protein of skeletal muscle thin filaments and probably acts as a ruler for the thin filament length. Cardiac muscles of higher vertebrates have been shown earlier to lack nebulin. Instead in human and chicken cardiac muscle the much smaller protein nebulette replaces nebulin.

The vasodilator-stimulated phosphoprotein promotes actin polymerisation through direct binding to monomeric actin.

The vasodilator-stimulated phosphoprotein (VASP) functions as a cellular regulator of actin dynamics. VASP may initialise actin polymerisation, suggesting a direct interaction with monomeric actin. The present study demonstrates that VASP directly binds to actin monomers and that complex formation depends on a conserved four amino acid motif in the EVH2 domain.

Ca-dependent binding of actin to gelsolin.

Ca(2+) of 0.3-1.0 microM induces both the exposure of tryptic cleavage sites within the gelsolin molecule inaccessible in the Ca-free conformation, and binding of one actin monomer to the N-terminal half of gelsolin.

Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle.

Kettin is a high molecular mass protein of insect muscle that in the sarcomeres binds to actin and alpha-actinin. To investigate kettin's functional role, we combined immunolabeling experiments with mechanical and biochemical studies on indirect flight muscle (IFM) myofibrils of Drosophila melanogaster. Micrographs of stretched IFM sarcomeres labeled with kettin antibodies revealed staining of the Z-disc periphery.

Sodium channel activity in leukemia cells is directly controlled by actin polymerization.

The actin cytoskeleton has been shown to be involved in the regulation of sodium-selective channels in non-excitable cells. However, the molecular mechanisms underlying the changes in channel function remain to be defined. In the present work, inside-out patch experiments were employed to elucidate the role of submembranous actin dynamics in the control of sodium channels in human myeloid leukemia K562 cells.

In vitro refolding of heterodimeric CapZ expressed in E. coli as inclusion body protein.

CapZ is a heterodimeric Ca(2+)-independent actin binding protein which plays an important role in organizing the actin filament lattice of cross-striated muscle cells. It caps the barbed end of actin filaments and promotes nucleation of actin polymerization, thereby regulating actin filament length. Here we report the expression of the two muscle-specific isoforms alpha2 and beta1, from chicken in Escherichia coli as individual subunits using the pQE60 expression vector and the subsequent renaturation of the functional CapZ heterodimer from inclusion bodies.

Identification and localisation of nebulin as a thin filament component of invertebrate chordate muscles.

The giant actin-binding protein nebulin is regarded as a component of the thin filaments in vertebrate skeletal muscles, whereas the existence of nebulin in invertebrate muscles has not yet been demonstrated. Using the cross-reactivities of polyclonal antibodies raised against nebulin from muscles of trout and lamprey, we were able to identify nebulin in the myofibrils of the cephalochordate Branchiostoma lanceolatum (lancelet) by immunoblot and immunofluorescence techniques. The approximately 720-kDa protein is localised in the I-bands of the sarcomere, where vertebrate nebulin has previously also been shown to be localised.

Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody.

Using a reconstituted complex of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2. As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187. In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch.

A six-module human nebulin fragment bundles actin filaments and induces actin polymerization.

We have investigated the interaction of a 6-repeat recombinant human nebulin fragment (S6R2R7) with F-actin, with Mg2+-induced actin paracrystals, and G-actin, respectively. This fragment corresponds to super-repeat 6, repeat 2 to 7 of human nebulin, and is located in the N-terminal part of the super-repeat region of the nebulin molecule. The S6R2R7 fragment included an immuno-tag of three amino-acid residues (EEF) at one end which was detectable by a monoclonal anti-tubulin YL1/2.

Conformational changes in actin induced by its interaction with gelsolin.

Actin cleaved by the protease from Escherichia coli A2 strain between Gly42 and Val43 (ECP-actin) is no longer polymerizable when it contains Ca2+ as a tightly bound cation, but polymerizes when Mg2+ is bound. We have investigated the interactions of gelsolin with this actin with regard to conformational changes in the actin molecule induced by the binding of gelsolin. ECP-(Ca)actin interacts with gelsolin in a manner similar to that in which it reacts with intact actin, and forms a stoichiometric 2:1 complex.

Actin-titin interaction in cardiac myofibrils: probing a physiological role.

The high stiffness of relaxed cardiac myofibrils is explainable mainly by the expression of a short-length titin (connectin), the giant elastic protein of the vertebrate myofibrillar cytoskeleton. However, additional molecular features could account for this high stiffness, such as interaction between titin and actin, which has previously been reported in vitro. To probe this finding for a possible physiological significance, isolated myofibrils from rat heart were subjected to selective removal of actin filaments by a calcium-independent gelsolin fragment, and the "passive" stiffness of the specimens was recorded.

Ca-dependent regulation of Na+-selective channels via actin cytoskeleton modification in leukemia cells.

With the use of the patch-clamp technique, physiological mechanisms of Na+ channel regulation involving submembranous actin rearrangements were examined in human myeloid leukemia K562 cells. We found that the actin-severing protein gelsolin applied to cytoplasmic surface of membrane fragments at a high level of [Ca2+]i (1 microM) increased drastically the activity of Na-selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca2+]i using the ionophore 4Br-A23187 also resulted in Na+ channel activation.

Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity.

We have investigated the cumulative effects of three smooth-muscle actin-binding proteins, gelsolin, caldesmon and tropomyosin, on actin activation of myosin Mg(2+)-ATPase activity under low-ionic-strength conditions. A combination of tropomyosin (at a stoicheiometric ratio to actin) and gelsolin (at a molar ratio to actin of up to 1:100) showed essentially additive stimulatory effects that were counteracted by caldesmon. Suppression of the gelsolin-induced activation of the ATPase by caldesmon was higher in the presence of tropomyosin although it was not complete even at stoicheiometric amounts of both proteins to actin.