3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Alteration of thyroidal responsiveness to TSH under the influence of circulating thyroid hormone: short feed-back regulatory effect.

In order to examine the hypothesis that the thyroidal responsiveness to TSH is under the influence of thyroid hormone, following the T3 injection to the mice, serum T3 concentrations and the response of thyroid tissue to a fixed dose of TSH in terms of intracellular colloid droplet formation was studied. The colloid droplets induced by TSH was significantly reduced when serum T3 was decreasing, while it was significantly increased when serum T3 was increasing. This results demonstrate for the first time the existence of short feed-back loop regulating intra-thyroidal function by circulating T3. To delineate the possible mechanism of action of T3, the thyroid gland of mouse whose serum T3 concentration was elevated by injecting 50 mug T3, was incubated with TSH in vitro. TSH-induced cyclic AMP generation was not inhibited at all but colloid droplet formation was significantly inhibited in the thyroid tissue of the animal whose serum T3 concentration was enormously high. Thus, it was demonstrated that the site at which T3 affects is beyond cylcic AMP generation but prior to endocytosis, being consistent with our previous results.     

Deficiency of subunits in heart mitochondrial NADH-ubiquinone oxidoreductase of a patient with mitochondrial encephalomyopathy and cardiomyopathy

The heart mitochondria isolated from a patient with hypertrophic cardiomyopathy associated with mitochondrial encephalomyopathy were analyzed by immunoblotting using specific antibody against each of the purified mitochondrial energy transducing complexes from beef heart. Subunits of NADH-ubiquinone oxidoreductase (Complex I) were markedly decreased and those of cytochrome c oxidase (Complex IV) were decreased to some extent, but the deficiency of any of these subunits was only partial. On the other hand, the contents of subunits of ubiquinol-cytochrome c oxidoreductase (Complex III) were normal. These results suggest that the decreased levels of some of the Complex I subunits might be the primary cause of disorder in this patient.     

An anti-carbohydrate monoclonal antibody inhibits cell-substratum adhesion of F9 embryonal carcinoma cells

A monoclonal rat IgM antibody (4C9) raised against F9 embryonal carcinoma cells reacted with fucosyl residues in poly-N-acetyllactosamine-type large carbohydrates of these cells (embryoglycan). The chemical properties and distribution of the antigen resembled those of SSEA-1. The monoclonal antibody was found to inhibit cell-substratum adhesion of F9 cells: in the presence of the antibody, cells grew as spherical cell aggregates on plastic dishes. When the antibody was added to the already spread cells, they displayed the initial sign of rounding up within 3 h; the rounding process was largely completed within 6 h. After removal of the antibody, cells resumed their normal morphology. The antibody could act in the presence of 2,4-dinitrophenol. In serum-free medium, F9 cells spread on plastic dishes coated with fibronectin or with laminin, and the process was also inhibited by the antibody. Immuno-electronmicroscopy revealed that 4C9 antigen was diffusely distributed over the cell surface of F9 cells. The distribution of the antigen was not altered generally after culturing with the antibody for 6 h. Another monoclonal rat IgM antibody, which did not react with embryoglycan and resembled anti-Forssman, did not inhibit cell-substratum adhesion of F9 cells, in spite of its reactivity to the cells. Thus, a glycoprotein with fucosyl (poly)-N-acetyllactosamine structure appears to be involved in cell-substratum adhesion of F9 cells.     

Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumoniae and Serratia marcescens

The synthesis of the OmpF porin in Escherichia coli K-12 was highly and reversibly inhibited by 5 mM salicylate in the bacterial growth medium, and salicylate also inhibited the OmpC porin synthesis, although only weakly. The full expression of the salicylate effect was presumed to require the ompB gene product on comparison between the wild type and ompB mutant strains. The salicylate effect was also observed for the porin protein synthesis in Klebsiella pneumoniae and Serratia marcescens, although an ompB-like gene remains to be identified in both species.     

Confocal laser microscopy of dystrophin localization in guinea pig skeletal muscle fibers

A confocal laser microscope was used to analyze the localization pattern of dystrophin along the sarcolemma in guinea pig skeletal muscle fibers. Hind leg muscles of the normal animals were freshly dissected and frozen for cryostat sections, which were then stained with a monoclonal antidystrophin antibody. In confocal laser microscopy, immunofluorescence staining in relatively thick sections could be sharply imaged in thin optical sections. When longitudinal and transverse sections of muscle fibers were examined, the immunostaining of dystrophin was seen as linearly aligned fluorescent dots or intermittent lines along the sarcolemma. In longitudinally cut muscle fibers, many fluorescent dots, but not all, corresponded to the sarcomere pattern, especially the I band. Sections cut tangential to the sarcolemma also showed a lattice-like pattern of longitudinal and transverse striations of fluorescent dots. Double staining for dystrophin and vinculin showed that the two proteins were not exactly colocalized. The end portions of muscle fibers were much more intensely stained with antidystrophin antibody than the central portions, following the contour of elaborate surface specializations at the myo-tendon junction. The staining pattern at the myo-tendon junction was also discontinuous. These confocal microscopic observations suggest that dystrophin may be localized in a nonuniform, discontinuous pattern along the sarcolemma and in some relationship with the underlying myofibrils.