Use of the fluorescent probe 1-anilinonaphthalene-8-sulfonate to study the synaptosomal transmembrane potential

The transmembrane potential (TMP) of synaptosomes isolated from the rat cerebral cortex was studied with the aid of 1-anilinonaphthalene-8-sulfonate (ANS). Addition of valinomycin to the synaptosomes was accompanied by an increase in the intensity of fluorescence of the probe at 464 nm (_exc = 365 nm), which is interpreted as reflecting hyperpolarization of the synaptosomal membranes. A decrease in the K⁺ gradient on the synaptosomal membrane (a decrease in TMP), achieved by preincubation of the synaptosomes with ouabain (1 mM) or an increase in the concentration of extrasynaptosomal K⁺ from 5 to 20 mM appreciably reduced the effect of valinomycin. Valinomycin had no effect on synaptosomes exposed to osmotic shock. It is suggested that valinomycin-induced changes in the intensity of fluorescence of ANS be used as a test of preservation of the K⁺ gradient (the presence of a TMP) by synaptosomal preparations, i.e., of the active state of the synaptosomes.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 3, pp. 300–302, March, 1980.     

Use of surface potential spectral characteristics for solving the inverse problem in electroneurography

The changes in the power spectra of single-fibre extracellular action potentials (SFEAPs) generated in an infinite anisotropic frequency-dependent volume conductor, which occurred as a result of alterations in the propagation velocity v and duration T_in of the intracellular action potential (IAP) were analytically determined. Effects of the temporal and spatial dispersions of almost synchronously activated fibres on the power spectrum of compound extracellular potentials (CEPs) were analysed for different shapes and sizes of the activated fibres' territory. It was found that, as a result of desynchronisation in the fibres' activation, dips existed in the CEP power spectra and that the frequencies of the dips depended on the degree of desynchronisation but did not depend on the velocity. It was shown that the hypothetical power spectrum of compound IAP was sensitive to the variations in the desynchronisation in the fibres' activation and in the risetime and duration of IAP even at a great fibre electrode distance typical for surface recordings.     

JP‐45/JSRP1 Variants Affect Skeletal Muscle Excitation–Contraction Coupling by Decreasing the Sensitivity of the Dihydropyridine Receptor

JP‐45 (also JP45; encoded by JSRP1) is an integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane interacting with Ca_v1.1 (the α.1 subunit of the voltage‐sensing dihydropyridine receptor, DHPR) and the luminal calcium‐binding protein calsequestrin. Two JSRP1 variants have been found in the human population: c.323C>T (p.P108L) in exon 5 and c.449G>C (p.G150A) in exon 6, but nothing is known concerning the incidence of these polymorphisms in the general population or in patients with neuromuscular diseases nor the impact of the polymorphisms on excitation–contraction (EC) coupling. In the present report, we investigated the frequencies of these two JSRP1 polymorphisms in the Swiss malignant hyperthermia population and studied the functional impact of the variants on EC coupling. Our results show that the polymorphisms are equally distributed among malignant hyperthermia negative, malignant hyperthermia equivocal, and malignant hyperthermia susceptible individuals. Interestingly, however, the presence of either one of these JP‐45 variants decreased the sensitivity of the DHPR to activation. The presence of a JSRP1 variant may explain the variable phenotype seen in patients with malignant hyperthermia carrying the same mutation and, more importantly, may counteract the hypersensitivity of EC coupling caused by mutations in the RYR1 gene.     

Biochemical characteristics of synaptosomes and mitochondria of the motor cortex after sensory deprivation

Changes in cytochrome oxidase, monoamine oxidase, acetylcholinesterase, and Na, K-ATPase activity following early light deprivation, were found in fractions of heavy and light synaptosomes and mitochondria isolated from the, bodies of neurons of the rat motor cortex by gradient centrifugation. These changes differed in direction for different metabolic cycles and were specific in individual ultrastructures of the cell. The effect of sensory impulsation on functional activity of neurons in the various cortical projection areas is discussed.Laboratory of Biohistochemistry, Brain Institute, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR N. K. Bogolepov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 10, pp. 424–426, October, 1978.     

Block of receptor response in the stretch receptor neuron of the crayfish by gadolinium

The trivalent lanthanide gadolinium was found to block the mechanotransducer response in the stretch receptor neuron of the crayfish. At normal calcium concentration (13.5 mm) a 50 per cent block of the receptor current was found at 395 ± 59 (mean ± SD), μM gadolinium. At a calcium concentration of 1.35 mm a 50 per cent block of the receptor current was obtained at 103 ± 14 (mean ± SD) μM gadolinium. The potential activated potassium current was also affected by gadolinium. At 200 μM the amplitude of the peak outward current as a result of a 90 mV positive potential step was decreased by about 40 per cent. The fast inward sodium current was decreased less than 10 per cent by gadolinium. It is concluded that in the crayfish stretch receptor gadolinium blocks the receptor current, reflecting block of stretch-activated channels, but at higher concentrations than have been described for other stretch-activated channels. In addition the outward rectifier potassium current is also blocked reflecting a block of potassium channels.     

Use of liposomes to studycAMP transport by lymphocyte membranes

Institute of Immunology, Ministry of Health of the USSR. (Presented by Academician of the Academy of Medical Sciences of the USSR A. D. Ado.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 3, pp. 246–248, March, 1990.     

Role of alpha chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor

Using anti-Tac (anti-alpha chain) and 2R-B (anti-beta chain) antibodies, we studied the roles of IL-2 receptor subunits (alpha and beta chains) in the formation of IL-2 and high-affinity IL-2 receptor complex, which is the initial event of IL-2 induced T cell growth. High-affinity IL-2 binding which was undetectable in the presence of 2R-B antibody at 4 degrees C became fully detectable when examined at 37 degrees C, which explained the lack of inhibition by 2R-B antibody of IL-2-induced proliferation of the cells expressing high-affinity IL-2 receptor. We further studied the mechanism of the 'reappearance' of high-affinity IL-2 binding in the presence of 2R-B antibody. The addition of IL-2 to the cells preincubated with radiolabeled or fluorescence-labeled 2R-B antibody resulted in a marked decrease in the antibody bound to the cells expressing high-affinity IL-2 receptor at 37 degrees C. This decrease was blocked by the presence of anti-Tac antibody, which inhibited IL-2 binding to alpha chain, but not by 7G7/B6 antibody, which recognized a non-IL-2 binding site of its chain. Furthermore, the decrease in cell-bound 2R-B antibody was not due to the internalization of beta chain-2R-B antibody complex, because the amount of cell-bound Mik-beta3 antibody recognizing a non-IL-2 binding epitope of beta chain remained unchanged, nor to the inhibition by simple competitive binding of IL-2 molecules to beta chain as judged from comparative studies of competitive binding inhibition. Taking these data together, the reappearance of high-affinity IL-2 binding was considered to be caused by the replacement of 2R-B antibody at the IL-2 binding site of beta chain by alpha chain-mediated IL-2, and it was strongly suggested that alpha chain-IL-2 complex has a key role in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor. alpha chain may function as a dimension converter of IL-2 to effectively deliver IL-2 molecules to a relatively small number of beta chains in the dynamics of the formation of high-affinity IL-2 binding in T cells.     

A new mechanism of action of vasopressin on neuron membranes

A. I. Gertsen Leningrad State Pedagogic Institute. Leningrad State University. (Presented by Academician of the Academy of Medical Sciences of the USSR G. N. Kryzhanovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 111, No. 2, pp. 115–116, February, 1991.     

Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis

A histidine substitution for the outermost arginine in II/S4 of the ₁ subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca current inactivation in myotubes from HypoPP patients by –40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle ₁, we introduced the corresponding mutation into the rabbit cardiac ₁ subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with and ₂ subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (–5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by –20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.     

A study of lectin bindings to the fetal membranes.

Human tissues contain carbohydrates for a main component, functioning as a source and reservoir of energy, connective and supporting element, recognition site and related tasks. Our main interest is to reveal the synthesis and distribution of carbohydrate elements in human fetal membranes. The aim of our work was to clarify, which kinds of elements containing carbohydrates, existed in the fetal membranes. Therefore we applied a lectin-binding study using the following FITC labelled lectins: ConA, WGA, PNA, LCA, RCA. This lead to the result, that ConA, LCA, WGA and RCA produced a positive reaction in the amnion epithelium, which was negative when using PNA. The basement membrane I showed an intense fluorescence when we used ConA, LCA and WGA, using RCA it was weaker and using PNA fluorescence was nearly missing. The examination of the amniotic fibroblast and intercellular substance showed a positive reaction with all lectins, but the intercellular substance lead to weaker fluorescence. The chorionic fibroblasts, intercellular substance and basement membrane II produced fluorescence using ConA, LCA, WGA and PNA, but no reaction could be examined, when using RCA. The trophoblastic cells did not react with LCA and RCA. The intercellular substance reacted positively with all lectins.     

Protein adsorption behaviour of ionogenic poly(HEMA) membranes: a fluorescence study

A series of ionogenic poly(HEMA) membranes which were prepared by bulk copolymerization of 2-hydroxyethylmethacrylate (HEMA) and anionic or cationic comonomers, acrylic acid (AA), and dimethylaminoethylmethacrylate (DMAEMA), were characterized by equilibrium swelling measurements, surface free energies, and protein adsorption studies. It was found that their equilibrium water content (EWC) values are greater than 40% which increases with increasing comonomer concentration. That is why the surface free energy is approximately the same (～60 erg cm^-2) for all surfaces and does not depend mainly on the composition of the polymer matrix. The adsorption of two plasma proteins that have received much attention, i.e. BSA and fibrinogen, on these membranes was followed by fluorimetric measurements as a function of time. The uptake of proteins from dilute solutions appeared to be directly related to the type and density of surface charge, and also structural properties of the proteins.     

Physical characteristics of nonimmunologic adherence of IgG to RBC ghost membranes

These studies were undertaken with the aim of detecting possible differences between immunologic binding of IgG antibodies to erythrocyte membranes and the well documented nonimmunologic adherence to erythrocytes of large numbers of IgG molecules. In the experiments described D⁺(Rho) erythrocyte ghosts were incubated with either ¹²⁵I labelled anti-D or ¹²⁵I IgG and then thoroughly washed. The ghosts which contained either immunologically bound anti-D or adherent IgG were then passed through a French press which converted the ghosts into microvesicles composed of membrane material. This procedure resulted in the selective removal of nonimmunologically adherent IgG from the microvesicles while leaving most of the antibody still firmly bound to the microvesicles. A series of control studies revealed that this observation was not limited to a single preparation of anti-D or IgG, and was not related to the number of IgG molecules attached to the erythrocyte membrane. Selective methods of chemical elution did not distinguish between the two forms of IgG attachment nor did another physical manipulation i.e. freezing and thawing. Separation of the sheared microvesicles into distinct classes indicated that antibody binding and nonimmunologic adherence of IgG molecules occurred equally to each of the different vesicle classes. These observations indicated that antibody binding and nonimmunologic adherence of IgG differ with regard to their physical characteristics and this difference may be a reflection of the inability of nonimmunologically adherent molecules to react with the Coombs'' reagent.