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Chionodracine (Cnd) is a 22-residue peptide of the piscidin family expressed in the gills of the Chionodraco hamatus as protection from bacterial infections. Here, we report the effects of synthetic Cnd on both Psychrobacter sp. TAD1 and Escherichia coli bacteria, as well as membrane models. We found that Cnd perforates the inner and outer membranes of Psychrobacter sp. TAD1, making discrete pores that cause the cellular content to leak out. Membrane disruption studies using intrinsic and extrinsic fluorescence spectroscopy revealed that Cnd behaves similarly to other piscidins, with comparable membrane partition coefficients. Membrane accessibility assays and structural studies using NMR in detergent micelles show that Cnd adopts a canonical topology of antimicrobial helical peptides, with the hydrophobic face toward the lipid environment and the hydrophilic face toward the bulk solvent. The analysis of Cnd free energy of binding to vesicles with different lipid contents indicates a preference for charged phospholipids and a more marked binding to native E. coli extracts. Taken with previous studies on piscidin-like peptides, we conclude that Cnd first adsorbs to the membrane, and then forms pores together with membrane fragmentation. Since Cnd has only marginal hemolytic activity, it constitutes a good template for developing new antimicrobial agents.

Steady state and time-resolved fluorescence studies on native, desulpho and deflavo xanthine oxidase (XO) have been carried out to investigate the conformational changes associated with the replacement of the molybdenum double bonded sulphur by oxygen and the removal of the flavin adenine dinucleotide (FAD). The steady state quenching experiments of the intrinsic tryptophan residues of the enzyme show that all the nine tryptophans are accessible to neutral quencher, acrylamide, in the native as well as desulpho and deflavo enzymes. However, the number of the tryptophan residues accessible to the ionic quenchers, potassium iodide and cesium chloride, increases upon removal of the FAD centre from the enzyme. This indicates that two tryptophan residues move out from the core of the enzyme to the solvent upon the removal of the FAD. The time-resolved fluorescence studies were carried out on the native, desulpho and deflavo XO by means of the time-correlated single photon counting technique, and the data were analysed by discrete exponential and maximum entropy methods. The results show that the fluorescence decay curve fitted best to a three-exponential model with lifetimes tau(1)=0.4, tau(2)=1.4 and tau(3)=3.0 ns for the native and desulpho XO, and tau(1)=0.7, tau(2)=1.7 and tau(3)=4.8 ns for the deflavo XO. The replacement of the molybdenum double bonded sulphur by oxygen in the desulpho enzyme does not cause any significant change of the lifetime components. However, removal of the FAD centre causes a significant change in the shortest and longest lifetime components indicating a conformational change in the deflavo XO possibly in the flavin domain. Decay-associated emission spectra at various emission wavelengths have been used to determine the origin of the lifetimes. The results show that tau(1) and tau(3) of the native and desulpho XO originate from the tryptophan residues which are completely or partially accessible to the solvent but tau(2) corresponds to those residues which are buried in the core of the enzyme and not exposed to the solvent. For deflavo enzyme, tau(2) is red shifted compared to the native enzyme indicating the movement of tryptophan residues from the core of the enzyme to the solvents.

The effects of methanol, acetone, and ethylene glycol (up to 50% v/v) on elementary steps in the reactions of horseradish peroxidase (HRP) and lactoperoxidase (LPO) were studied by means of the stopped-flow method and the difference spectrum. The rate constant (k3,app) of the oxidation reaction of p-cresol with HRP compound II was remarkably reduced in the presence of organic solvents (to 2.3%, 1.8% and 9.4% of the original value in the presence of 50% (v/v) of methanol, acetone and ethylene glycol, respectively), then to a lesser degree were decreased the rate of the oxidation reaction with LPO compound II, and the rate of the oxidation reaction with HRP compound I. These reductions in the reaction rates were not due to competitive inhibition of the solvents, but considered to be related to the degree of exposure of the electron transfer route to the medium. While the rate constant of compound I formation (k1,app) was moderately affected by organic solvents in the case of HRP, the reaction rate with LPO was scarcely affected by organic solvents, being in harmony with the compact heme crevice which probably hampers penetration of solvent molecules. The rate constant (k2,i,app) of the oxidation reaction of an iodide ion by HRP compound I was also hardly affected by the solvents. On the basis of these facts, the mechanism of electron transfer from donors to compound I and compound II is discussed.

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, α-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.

Saccharomyces cerevisiae NCYC 239 suspended in solutions of NaCl showed two distinct plateaus in plots of electrophoretic mobility vs. pH, corresponding to p K a values of approx. 2 and 5. This is in contrast to cells suspended in buffer where only a single p K a (4) can be determined. Modification of cells with KI/I 2 or nitrous acid led to altered electrophoretic mobility, indicating the presence of sulphydryl and amino groups, respectively, in the yeast cell surface, whereas uranyl nitrate modification had little effect, suggesting phosphate groups to be absent. Electron micrographs showed visible effects of KI/I 2 and nitrous acid modification on cell membrane structure, and in these modified cells amphotericin B uptake was rapid. It is suggested that diffusion through the cell wall is the rate-limiting step for amphotericin B uptake. An activation energy of 20 kJ·mol −1 was determined for uptake of amphotericin B by unmodified cells.

The transformation of arachidonic acid by the rat thyroid in vitro has been investigated. At least two metabolites have been partially characterized: they differed from known metabolites of arachidonic acid in terms of retention volume in liquid chromatography, ultraviolet spectrophotometry and pharmacology (formation not inhibited by indomethacin and enhanced by eicosatetraynoic acid). The analysis by chemical ionization mass spectrometry suggested that these metabolites might be diketo-monohydroxy- and monoketo-dihydroxy-compounds. The conversion of arachidonic acid into these compounds was stimulated by ionophore A23187, decreased by the peroxidase inhibitor methimazole and potentiated by iodide, suggesting that this pathway is under the control of Ca2+ and of a peroxidase product.

1. The regulation of protein synthesis in the rat-thyroid gland was studied utilizing both a tissue slice system and a cell-free amino acid incorporating system. 2. Thyroid tissue slices and thyroid cell-free amino acid incorporating systems obtained from iodine-deficient rats both had an increased ability to incorporate [14C]valine into protein compared to similar systems obtained from control rats. 3. The addition of low concentrations of thyroxine and iodide to the incubation medium reduced the increased incorporation observed in iodine-deficient rat thyroid glands to levels near those of controls in both experimental systems. 4. It was observed that the increased ability for [14C]valine incorporation in iodine-deficient rat-thyroid glands and the inhibitory action of thyroxine and iodide were associated with the microsomal fraction. 5. The action of thyroxine and iodide is discussed in terms of the regulation of protein synthesis. It is suggested that thyroxine and iodide act as ribosomal inhibitors of protein synthesis in the rat-thyroid gland.

An in vitro system is described in which iron incorporation into rabbit reticulocytes and bone marrow cells from pure transferrin is qualitatively comparable to that found in vivo. The amount of stromal-bound iron reproducibly represents 2% and 12% of the total iron incorporated respectively into reticulocytes and bone marrow cells after 2.5 h of incubation. After 60 min of incubation only 40% of the reticulocyte stromal-bound iron behaved like a metabolic intermediate. All the metabolically active, membrane-bound iron could be accounted for a transferrin iron. Of the transferrin found in washed cells, 50–60% was recovered in the cytoplasmic fraciton, and was shown by isoelectric focusing to be undergraded. It is likely that it represents intracellular transferrin. The rate of hemoglobin synthesis was linear for at least 2.5 h in both reticulocytes and bone marrow cells, while bone marrow cells showed z greater iron incorporation into heme than reticulocytes. There was no detectabel low molecular weight iron in the cell supernatant of reticulocytes or bone marrow cells and hemoglobin-iron accounted for more than 92% of the total iron incorporated into the reticulocytes.

1. 1. F-actin component isolated directly from myofibrils, i.e. “natural F-actin”, slowly loses its flow birefringence in 0.1 M KCl. 2. 2. Various nucleotides have a protective effect on the birefringence properties of natural F-actin; ATP or ADP together with Mg 2+ was most effective. 3. 3. Acetone treatment converted natural F-actin into F-actin of Straub type. 4. 4. When treated with 0.6 M KI solution, natural F-actin showed the properties of F-actin which was prepared with KI from myofibrils. 5. 5. Repeated homogenization of precipitated natural F-actin in 0.1 M KCl caused little change in properties, but repeated depolymerization and repolymerization converted natural F-actin gradually into Straub F-actin. 6. 6. When natural F-actin was partially digested with trypsin, it became similar to Straub F-actin, and repolymerization after depolymerization converted it completely into Straub F-actin.