C-terminal actin-binding sites of smooth muscle caldesmon switch actin between conformational states

Caldesmon is a component of the thin filaments of smooth muscles where it is believed to play an essential role in regulating the thin filaments’ interaction with myosin and hence contractility. We studied the effects of caldesmon and two recombinant fragments CaDH1 (residues 506–793) and CaDH2 (residues 683–767) on the structure of actin–tropomyosin by making measurements of the fluorescence polarisation of probes specifically attached to actin. CaDH1, like the parent molecule caldesmon, is an inhibitor of actin–tropomyosin interaction with myosin whilst CaDH2 is an activator. The F-actin in permeabilised and myosin free rabbit skeletal muscle ‘ghost’ fibres was labelled by tetramethyl rhodamine-isothiocyanate (TRITC)–phalloidin or fluorescein-5′-isothiocyanate (FITC) at lysine 61. Fluorescence polarisation measurements were made and the parameters Φ_A, Φ_E, Θ_1/2 and N were calculated. Φ_A and Φ_E are angles between the fiber axis and the absorption and emission dipoles, respectively; Θ_1/2 is the angle between the F-actin filament axis and the fiber axis; N is the relative number of randomly oriented fluorophores. Actin–tropomyosin interaction with myosin subfragment-1 induced changes in the parameters of the polarised fluorescence that are typical of strong binding of myosin to actin and of the ‘on’ conformational state of actin. Caldesmon and CaDH1 (as well as troponin in the absence of Ca²⁺) diminished the effect of S-1, whereas CaDH2 (as well as troponin in the presence of Ca²⁺) enhanced the effect of S1. Thus the structural evidence correlates with biochemical evidence that C-terminal actin-binding sites of caldesmon can modulate the structural transition of actin monomers between ‘off’ (caldesmon and CaDH1) and ‘on’ (S-1 and CaDH2) states in a manner analogous to troponin.     

Artificially applied tensions normalize development of relaxed Xenopus Laevis embryos

Relaxation of tensions of the surface of Xenopus laevis embryos at the late blastula stage leads to deep and diverse developmental defects and increased variability in mutual position and volume ratios of the axial rudiments. Here, we demonstrate that the development of such embryos was markedly normalized if the relaxed tensions were restored in one of two ways: (1) isotropic stretching of the blastocoel roof induced by incubation of relaxed embryos in a hypotonic medium or (2) anisotropic stretching of embryos on two needles. In the latter case, we succeeded in restoring the morphological axis not only after longitudinal stretching, but also after transverse stretching, and the new axis had signs of anteroposterior polarity. The role of isotropic and anisotropic tensions in organization of the early amphibian development is discussed.     

EEG Spectral Characteristics in Junior Schoolchildren with Learning Problems

EEG spectral characteristics were studied in two age groups (7–8.5 and 8.5–10 years) of mentally healthy children and children with learning problems at rest and during performance of a Raven test. It was shown that slow frequencies are more pronounced in the EEG of 7- to 8.5-year-old children with learning problems than in EEG of healthy children of the same age group. An immature form of EEG activation, i.e., an increase not only in the but also in the frequencies during activity, was characteristic of these children. The reaction of the activation of the definitive type develops between the 8.5–10 years of age. This reaction is correlated with an increase in the efficiency of the sensory perceptive and sensorimotor activity. The distinctive feature of children with learning problems between 8.5–10 years of age is a greater expression of slow frequencies in the baseline EEG of the frontal (in particular, left frontal) areas of the cortex. The obtained results are considered as a reflection of a retardation of the functional maturation of the brain structures responsible for the deficit of involuntary and voluntary attention and the disorder of a systemic organization of perception and analytical–synthetic brain activity as compared to the normal age characteristics. Possible neurophysiological mechanisms responsible for learning problems in junior schoolchildren are discussed on the basis of the obtained results and evidence from the literature.     

Synthesis of [26-2H(3)]brassinosteroids

A number of [26-2H(3)]brassinosteroids were prepared for biochemical studies. The parent, nondeuterated compounds were considered to be biosynthetic intermediates in brassinosteroid biosynthesis. Claisen rearrangement was used to construct the steroidal side chain. Deuterium was introduced by reducing the corresponding intermediates with lithium aluminium deuteride.     

Embryo recovery and pregnancy rates after the delay of ovulation and fixed time insemination in superstimulated beef cows

The aim of this study was to evaluate the effect of delaying ovulation subsequent to superstimulation of follicular growth in beef cows (Bos indicus) on embryo recovery rates and the capacity of embryos to establish pregnancies. Ovulation was delayed by three treatments using either progesterone (CIDR-B) or a GnRH agonist (deslorelin). Multiparous Nelore cows (n = 24) received three of four superstimulation treatments in an incomplete block design (n = 18 per group). Cows in Groups CTRL, P48 and P60 were treated with a CIDR-B device plus estradiol benzoate (EB, 4 mg, i.m.) on Day-5, while cows in Group D60 were implanted with deslorelin on Day-7. Cows were superstimulated with FSH (Folltropin-V, 200 mg), from Day 0 to 3, using twice daily injections in decreasing amounts. All cows were treated with a luteolytic dose of prostaglandin on Day 2 (08:00 h). CIDR-B devices were removed as follows: Group CTRL, Day 2 (20:00 h); Group P48, Day 4 (08:00 h); Group P60, Day 4 (20:00 h). Cows in Group CTRL were inseminated at 10, 20 and 30 h after first detected estrus. Ovulation was induced for cows in Group P48 (Day 4, 08:00 h) and Groups P60 and D60 (Day 4, 20:00 h) by injection of LH (Lutropin, 25 mg, i.m.), and these cows were inseminated 10 and 20 h after treatment with LH. Embryos were recovered on Days 11 or 12, graded and transferred to synchronized recipients. Pregnancies were determined by ultrasonography around Day 100. Data were analyzed by mixed procedure, Kruskal-Wallis and Chi-square tests. The number of ova/embryos, transferable embryos (mean +/- SEM) and pregnancy rates (%) were as follows, respectively: Group CTRL (10.8+/-1.8, 6.1+/-1.3, 51.5), P48 (12.6+/-1.9, 7.1+/-1.0, 52.3), P60 (10.5+/-1.6, 5.7+/-1.3, 40.0) and D60 (10.3+/-1.7, 5.0+/-1.2, 50.0). There were no significant differences among the groups (P > 0.05). It was concluded that fixed time AI in association with induced ovulation did not influence embryo recovery. Furthermore, pregnancy rates in embryos recovered from cows with delayed ovulation were similar to those in embryos obtained from cows treated with a conventional superstimulation protocol.     

Epithelial barrier function: assembly and structural features of the cornified cell envelope

Terminally differentiating stratified squamous epithelial cells assemble a specialized protective barrier structure on their periphery termed the cornified cell envelope (CE). It is composed of numerous structural proteins that become cross-linked by several transglutaminase enzymes into an insoluble macromolecular assembly. Several proteins are involved in the initial stages of CE assembly, but only certain proteins from a choice of more than 20 different proteins are used in the final stages of CE reinforcement, apparently to meet tissue-specific requirements. In addition, a variable selection of proteins may be upregulated in response to genetic defects of one of the CE proteins or tissue injury, in an effort to maintain an effective barrier. Additionally, in the epidermis and hair fiber cuticle, a layer of lipids is covalently attached to the proteins, which provides essential water barrier properties. Here we describe our current understanding of CE structure, a possible mechanism of its assembly, and various disorders that cause a defective barrier.     

Development and applications of surface plasmon resonance-based von Willebrand factor-collagen binding assay

Von Willebrand factor (vWf) functions both as a carrier of factor VIII (fVIII) in plasma and as an adhesive protein providing the primary link between collagen of the extracellular matrix and platelets sequestered from blood flow. The functional activity of vWf correlates with the level of its binding to collagen, which is commonly measured in the enzyme-linked immunosorbent assay (ELISA). We developed an automated collagen-binding assay employing the surface plasmon resonance (SPR) phenomenon, which allows one to quantitatively measure the binding of purified vWf and vWf-containing therapeutic fVIII concentrates to collagen type III immobilized on a biosensor chip. The results of the SPR-based assay highly correlated (r = 0.987) with collagen-binding ELISA. The advantages of the SPR-based assay are its higher accuracy and reproducibility in comparison with ELISA. We applied the developed assay for monitoring structural changes in the vWf component of plasma-derived fVIII/vWf concentrates during a virus inactivation procedure performed by heat treatment. We determined the critical residual moisture content of 2% that can be present in lyophilized concentrates during heat-treatment procedures without causing deteriorative changes in vWf properties. Our data suggest that the SPR-based assay is a useful tool in the development of industrial virus-inactivation procedures, allowing one to preserve vWf activity and achieve the maximal therapeutic efficacy of fVIII/vWf concentrates.     

Massive light-driven translocation of transducin between the two major compartments of rod cells: a novel mechanism of light adaptation

We report a new cellular mechanism of rod photoreceptor adaptation in vivo, which is triggered by daylight levels of illumination. The mechanism involves a massive light-dependent translocation of the photoreceptor-specific G protein, transducin, between the functional compartments of rods. To characterize the mechanism, we developed a novel technique that combines serial tangential cryodissection of the rat retina with Western blot analysis of protein distribution in the sections. Up to 90% of transducin translocates from rod outer segments to other cellular compartments on the time scale of tens of minutes. The reduction in the transducin content of the rod outer segments is accompanied by a corresponding reduction in the amplification of the rod photoresponse, allowing rods to operate in illumination up to 10-fold higher than would otherwise be possible.     

Correlation between HSP90 induction kinetics in murine leukemia cells and the amount of cisplatin over a wide range of cytostatic concentrations

The induction of HSP90 in murine erythroleukemia cells, clone F4 N, by cisplatin (DDP) was examined using indirect immunofluorescence and avidin-biotin technique, and compared with cisplatin cytotoxicity. A reverse dependence of HSP90 induction time was found on a wide range of cisplatin concentrations (0.5-10 microM), which proved to be cytostatic up to 48 h of continuous treatment. Thus, the observed induction pattern of HSP90 in F4 N cells strictly correlated with their high tolerance toward DDP. This indicates that HSP90 might be responsible, at least in part, for cisplatin resistance of F4 N cells.