Flavin-photosensitized oxidation of reduced c-type cytochromes. Reaction mechanism and comparison with photoreduction of oxidized cytochromes by flavin semiquinones

In order to compare the oxidation and reduction reactions of c-type cytochromes (cytochrome c552 from the green alga Monoraphidium braunii and horse heart cytochrome c) by different flavins (lumiflavin, riboflavin and FMN), laser flash photolysis studies have been carried out using either reduced or oxidized protein in the presence of triplet or semiquinone flavin, respectively. The reaction kinetics clearly demonstrate that cytochrome oxidation is mediated by the flavin triplet state. The rate constants for reduction are 20-100 times smaller than those for oxidation, indicating that the triplet state is a more effective reactant than is the semiquinone. This is attributed to its excited state nature and correspondingly high free energy content. The rate constants for both the reduction and oxidation of cytochrome c552 by riboflavin are significantly smaller than those obtained with lumiflavin, suggesting a steric interference of the ribityl side chain in the flavin-cytochrome interaction. The comparison between oxidation and reduction indicates that the former process is less affected by steric hindrance than the latter. Both reduction and oxidation of cytochrome c552 by FMN show an ionic strength dependence with the same sign, consistent with a negatively charged reaction site on the cytochrome. The magnitude of the electrostatic effect is slightly smaller for reduction than it is for oxidation. A pattern quite similar to that observed with cytochrome c552 was obtained when parallel experiments were carried out with horse cytochrome c, although differences were observed in the steric and electrostatic properties of the electron transfer site(s) in these two cytochromes. These results suggest that the same or closely adjacent sites on the proteins are involved in the oxidation and reduction reactions. The biochemical implications of this are discussed.     

Positive residues involved in the voltage-gating of the mitochondrial porin-channel are localized in the external moiety of the pore.

The role of positive charges located on the hydrophilic surface of the mitochondrial outer membrane channel was investigated by studying the interaction between LDAO-solubilized porin and a cation-exchanger column. The binding of porin to the column material was inhibited when the elution buffer had a pH of 9 or when 2 mM dextran sulfate was added to the buffer at neutral pH. Interestingly, the addition of a synthetic copolymer of methacrylate, maleate and styrene known as a potent modulator of the voltage-dependence, did not influence the interaction between column material and porin. Incubation of porin with fluorescein isothiocyanate (FITC) resulted in the isolation of a porin fraction in which on average two lysines located on the surface of the pore-forming complex per 35 kDa polypeptide were modified. The voltage-dependence of the fluorescein isothiocyanate modified porin was strongly decreased as compared with the unmodified porin. The experiments presented here give the first biochemical evidence that positively charged lysine residues located on the surface of the channel-forming complex are responsible for the gating of the mitochondrial porin-channel.     

On predatory tendencies in the feeding ecology of the fairy shrimp Streptocephalus proboscideus (Frauenfeld, 1873) (Crustacea: Anostraca)

The feeding habits of the filter-feeding fairy shrimp Streptocephalus proboscideus are documented experimentally by offering them ciliates, Volvox, rotifers, nematodes and small crustaceans as prey. Escape capabilities (e.g. swimming speed) rather than size or shape were found to determine these animals' vulnerability to predation by the fairy shrimp. Ingestion rates for Volvox increased hyperbolically with size and, at the high temperatures in which they live, fairy shrimps may daily remove the equivalent of their body weight from the environment.     

Use of synthetic resin cases for the scanning electron microscopic study of the kidney tubule system

Aim of our present work was to investigate a new method to study the three-dimensional arrangement, the length and the diameter of the different parts of the renal tubules. The ureter was cannulated after blocking the urinary flow with a binding of the ureter itself at its intermediate third, and injected in it against flow a synthetic resin (Mercox) normally used for vascular corrosion casts. It was demonstrated that the binding maintained only for 24 hours is adequate for morphological studies of the urinary tracts from papillar ducts until the Henle's loop. On the contrary the binding maintained for 7 days induced marked changes in the tubular architecture similar to the first anatomo-pathological changes of the nephrosclerosis following a chronic obstructive nephropathy.     

Tumor necrosis factor antagonizes inhibitory effect of azidothymidine on human immunodeficiency virus (HIV) replication in vitro.

Tumor necrosis factor alpha (TNF-alpha) completely reverses the activity of azidothymidine (AZT) against human immunodeficiency virus type 1 (HIV-1) in MOLT-4 cell cultures. The 50% effective concentration of AZT, required to protect MOLT-4 cells against the cytopathic effect of HIV-1, increased from 5.8 nM in the absence of TNF-alpha to greater than 125 microM in the presence of TNF-alpha (100 U/ml). TNF-alpha also antagonized the anti-HIV-1 activity of dideoxycytidine but did not markedly affect the anti-HIV-1 activity of dextran sulfate. The intracellular phosphorylation pattern of AZT was not changed upon the presence of TNF-alpha.     

Validity of the nearest-neighbor approximation in the evaluation of the electrophoretic manifestations of DNA curvature

The validity of a theoretical nearest-neighbor model based on conformational energy calculations, for translating the deterministic fluctuations of DNA base sequences in superstructural elements, is tested on the electrophoretic data of 450 multimeric, duplex oligonucleotides with different sequence, periodicity, and complexity, corresponding to all the experimental data so far published. An accurate semiempirical linear relation between the gel electrophoretic retardation and the dispersion of the theoretical curvature was found which allows a prediction of electrophoretic anomalies of DNA tracts, e.g., the cyclic permutation gel assays, with great confidence. Such a pattern of agreement allows the interpretation of the gel electrophoresis retardation as due to an increase of activation energy required in straightening the DNA axis.