Water-insoluble protein fractions increase in the brain cortical tissue and liver of rats during aging in both sexes. This suggests a possible increase in the cross-linking of proteins which may be due to the formation of, for example, hydroxyl free radicals during several metabolic processes. In vivo application of centrophenoxine causes a reversal of this phenomenon in old rats. In vitro experiments show that the generation of hydroxyl free radicals by chemical systems like homolysis of H2O2 by redox coupling with Fe2+ leads to Fe3+ conversion, results in the cross-linking of bovine serum albumin and the mixed proteins of liver or brain homogenates of young rats. The cross-linked proteins have a very much increased molecular weight, they become mostly insoluble even in 6 M urea. Dimethylaminoethanol, the effective part of the centrophenoxine molecules, is able to diminish the extent of cross-linking, acting most probably as a free-radical scavenger. The results are discussed in terms of the "membrane hypothesis of aging". A molecular basis is proposed for the anti-aging effect of centrophenoxine.

Aging↗

PubMedDate not supplied

Results of space experiment program "Interferon".

The results of the biological space experiment "Interferon" performed by two international cosmonaut teams (26 May 1980, and 16 May 1981) aboard space laboratory Solyut-6 are reported: (1) Human lymphocytes separated from blood of healthy donors and placed into "Interferon I" equipment could be kept for 7 days in suspension culture under spaceflight conditions. Interferon production could be induced in human lymphocytes by preparations of different origin: virus, synthetic polyribonucleotides, bacterial protein and plant pigment. An increased lymphocyte interferon production in space laboratory compared to ground control was observed. (2) Human interferon preparations and interferon inducers placed in space laboratory at room temperature for 7 days maintained their biological activity. (3) A decrease of induced interferon production and natural killer activity of lymphocytes isolated from peripheral blood of cosmonauts was observed on the 1st day on Earth after 7-days spaceflight.

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PubMedDate not supplied

In vitro studies on the OH* and O2(-*) free radical scavenger properties of idebenone in chemical systems.

OH(*) free radicals were generated by Fenton reaction in the presence of bovine serum albumin (BSA). The decreasing water-solubility of BSA with increasing Fe(2+) concentrations of the system is a sensitive indicator of the cross-linking effects of the OH(*) free radicals. Idebenone (oxidized form) was solubilized for this experiment in DMSO and added to the system in final concentrations of 0.01 or 0.1%. Neither of these concentrations displayed any protective effect against the insolubilization of BSA. Therefore, oxidized idebenone has to be considered as a substance which reacts with OH(*) free radicals slower than the BSA itself, i.e., its oxidized form is not an efficient scavenger of this type of free radicals under the given circumstances. The ability of idebenone to scavenge superoxide radicals was tested in ( [Formula: see text] ) the pyrogallol system; and (ii) the xanthine-xanthine oxidase-nitro blue tetrazolium (XXO-NBT) system. Idebenone did not show any O(2)(-*) radical scavenging ability as revealed by these two in vitro methods, in the concentration ranges studied (up to 75 or 220 microg/ml, respectively). On the contrary, an increasing O(2)(-*) radical generation was observed with increasing concentrations of the drug in both test systems used. The possible biological significance of these observations is discussed in the light of other results like ESR spin trapping and measurements of superoxide dismutase (SOD) activity in various tissues.

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PubMedDate not supplied

Effects of idebenone on the composition of brain synaptosomal membranes of rats.

Male and female CFY rats of 21 months of age were treated per os by 50 mg idebenone per kg body weight/day for 5 weeks while the controls received only the solvent. Synaptosomal fractions of the brain cortex were prepared and tested as follows: (1) Labeling by 1,6-diphenyl-1,3,5-hexatriene (DPH) and the fluorescence anisotropy (r) of this label was measured at various optical densities. The average values of r extrapolated to zero optical density were 0.1831 +/- 0.0025 in the control group and 0.1854 +/- 0.0019 in the idebenone-treated animals; the difference is statistically not significant. (2) Labeling with 3 different spin labels (5-NS, N-oxyl-4', 4'-dimethyloxazolidine derivative of stearic acid with the doxyl groups in C-5; 16-NS, the same derivative of stearic acid with the doxyl groups in C-16 position; and MAL-SL, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxy). Routine ESR spectroscopy was applied to characterize the molecular motion of these labels in their microenvironment. These spin labels did not display any significant change in their environment under the influence of idebenone treatment. (3) Protein contents of the samples were measured and samples of synaptosomes containing 3.5 mg protein were dissolved in 1% Na(Dod)SO4 + 1% beta-mercaptoethanol and boiled for 2 min, then elaborated by gel filtration chromatography on a column of sepharose 2B. The elution fluid was collected in separate fractions of 1 ml volume and their protein content was estimated by measuring the absorbance at 280 nm. Distribution histogram of the protein fractions revealed no difference between the placebo- and verum-treated animals. These studies demonstrate that in vivo application of idebenone does not alter the composition of synaptosomal membranes.

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PubMedDate not supplied

The effect of idebenone on the total content and solubility characteristics of proteins and osmometric behavior of the intracellular mass in brain of CFY and SHRsp rats.

Female CFY rats (21 months old) and male spontaneously hypertensive, stroke-prone (SHRsp) rats (3 months old), in conventional housing conditions, received placebo (5% gum arabic solution) or 50 mg/kg bw/day idebenone suspended in 5% gum arabic, through a gastric tube for 5 weeks; then their brains were elaborated as follows: (1) Total proteins as well as water-soluble and water-insoluble proteins (WSP and WIP, respectively) were separated from the brain homogenate by centrifugation at 500 X g. The WIP fractions were tested also in vitro by heat denaturation at 64 degrees C (10 min) and by 3 M urea treatment. In the placebo group of CFY rats the total protein content was 113.9 mg per g fresh weight. WIP amounted to 27.2% of the total proteins. Idebenone-treatment did not alter the protein composition in these old rats. In the SHRsp rats the total protein content of the brain cortex was almost identical with that of the normal, Wistar-derived CFY rats of much more advanced age (about 2 years). The idebenone-treatment did not alter the protein content of the brain cortex, although the WIP content and the heat-resistant fraction of it increased significantly in this strain. (2) The osmotic potential of brain tissue was determined by measuring swelling or shrinkage velocities in Ringer solution, the osmotic concentration of which was rendered hypo- or hyperosmotic by dilution or addition of polythylene glycol (PEG 6000), respectively. Idebenone treatment exerted no effect on the osmometric properties of the brain tissue in either the normal old or the SHRsp rats.

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PubMedDate not supplied

The effects of idebenone on the superoxide dismutase, catalase and glutathione peroxidase activities in liver and brain homogenates, as well as in brain synaptosomal and mitochondrial fractions.

The cerebral cortex and liver of 20 female CFY rats of 21 months of age (at killing), kept in conventional housing conditions, were studied. Ten animals received placebo (5% gum arabic solution) and 10 other rats received 50 mg/kg body weight/day idebenone suspended in the gum arabic, through a gastric tube for 5 weeks (except Sundays). Liver and brain homogenates were obtained by homogenization of tissue pieces (10 mg/ml) in 0.32 M sucrose solution. Synaptosomal and mitochondrial fractions of the brain cortex were prepared by using established methods of density gradient centrifugation, pooling together the brains of two animals for each experiment. Protein contents of the samples were measured by means of a modified folin-phenol method. Superoxide dismutase (SOD), catalase (CA) and glutathione peroxidase (GP) activities were measured by using established biochemical methods. The activities were compared in the placebo and verum groups on a protein weight basis. The SOD activity increased slightly (but not significantly) in the total brain homogenate, whereas both the mitochondrial and synaptosomal fractions of the cortex displayed significant increases of this enzyme activity (28.5 and 16.3% respectively). SOD and CA activities decreased in the liver homogenate and all other parameters remained invariate both in the brain and its subfractions, except CA in the synaptosomal fraction where a slight decrease occurred (-15.6%).

Journal Article↗

PubMedDate not supplied

An in vitro model of aging: the influence of increasing physical density on enzyme activities of trypsin, xanthine oxidase and superoxide dismutase.

The enzyme activities of trypsin (using an artificial substrate, Nalpha-benzoyl-L-arginine-ethylester = BAEE), xanthine oxidase (XOD) and superoxide dismutase (SOD) were measured in the absence and presence of various concentrations of the following inert, water-soluble polymer viscogens: polyvinylpyrrolidone (PVP-40), polyethyleneglycol (PEG-6000) and bovine serum albumin (BSA). Enzyme activities measured in the absence of viscogens were taken as 100%. In the presence of the viscogens, enzyme activities decreased considerably as follows: (i) Trypsin: to 2 or 12% in reaction mixtures containing 64 mg/ml PVP-40 or 481 mg/ml PEG-6000, respectively. (ii) XOD: to 29.3% in a reaction mixture containing 116 mg/ml PVP-40, to 68.9% in a medium containing 266 mg/ml PEG-6000, and 38.1% in the presence of 138 mg/ml BSA. (iii) SOD: to 40.0, 19.9 and 16.6% in the same media as listed for XOD, respectively. The observations are consistent with the predictions of the molecular enzyme kinetic model (MEKM), and are also of importance for the membrane hypothesis of aging, since the latter explains the loss of cell functions by an age-dependent increase of intracellular density which may cause serious enzyme inhibitions.

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PubMedDate not supplied

Age-dependence of free radical-induced oxidative damage in ischemic-reperfused rat heart.

Oxygen free radical-induced oxidative damage is involved in both aging and ischemia-reperfusion. The purpose of this study was to determine the aging-induced oxidative alterations in rat heart as well as the age-dependence of heart injury following ischemia-reperfusion. A comparative study was performed on young and old ischemic-reperfused rat hearts. Protein oxidation and the ascorbyl radical level in heart tissue were determined in order to characterize the oxidative stress. Comparing the control conditions, old hearts have 31% more oxidized proteins as measured by protein carbonyl content, and 18% lower ascorbyl radical level as determined by ESR, than young ones. The extent of increase of protein oxidation and ascorbyl free radical depletion induced by ischemia-reperfusion is less pronounced in the old hearts (7 and 8% respectively), as compared to the young ones (55 and 21% respectively). Pre-treatment with a free radical scavenger, such as centrophenoxine, diminished the ischemia-reperfusion injury in both young and old rat hearts.

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PubMedDate not supplied

Alpha lipoic acid (ALA) protects proteins against the hydroxyl free radical-induced alterations: rationale for its geriatric topical application.

The well known OH* free radical scavenging properties of alpha-lipoic acid (ALA) cannot be easily utilized for biological experiments, because the compound is practically insoluble in water. We elaborated a simple method of preparing its Na-salt (Na-ALA) which proved to be water soluble. It has been demonstrated by ESR spin trapping experiments with DMPO, using the Fenton reaction as the source of OH* free radicals that Na-ALA maintains its OH* free radical scavenging ability: it reacts nearly an order of magnitude faster with these radicals than the spin trap itself. It was tested in two different systems to determine whether Na-ALA was able to protect bovine serum albumin (BSA) against the OH* free radical-induced polymerization and protein oxidation. (i) OH* free radicals were generated by Fenton reaction in the presence of BSA. This protein is polymerized by these radicals shown by the loss of its water solubility; Na-ALA exerted a considerable protective effect against this type of protein damage. (ii) BSA oxidation was induced by Co-gamma irradiation of 80 krad, resulting in a strong increase in the protein carbonyl content. Na-ALA inhibited this carbonyl formation very efficiently. The data suggest that the interaction of the OH radical with Na-ALA takes place on the disulfide group, yielding thiosulfinate or thiosulfonate. The results indicate that the geriatric topical application of Na-ALA may have an established rationale.

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PubMedDate not supplied

Inhibition of phospholipase C by U-73122 blocks one component of the receptor current in Limulus photoreceptor.

In the ventral nerve photoreceptor of Limulus a short, intense flash evokes a receptor current consisting of three components. In contrast to other hypotheses, we suggested previously that only the second component of the current is activated by the phospholipase C pathway, which releases calcium from intracellular stores by inositol trisphosphate. The present paper gives further evidence to our suggestion. It is demonstrated that U-73122, a specific inhibitor for the phospholipase C, selectively blocks the second-component. The first and third components are moderately affected and could still be activated after the complete block of the second one. Results support the idea that the first and third components are activated by pathways operating independently of phospholipase C.

Animals↗

PubMedDate not supplied

Photoreceptor cells with unusual functional properties on the ventral nerve of Limulus.

Normal photoreceptor cells on the ventral nerve of Limulus respond to a moderately intense flash with a large receptor potential or current. Occasionally, cells are found in which the same flash evokes only a small receptor potential or current. Our investigations reveal physiological reasons for the poor light sensitivity in these "unusual cells." In unusual cells prolonged illumination with intense light evokes a step-like inward current with an amplitude of some nanoamperes, but without a large transient peak. The current appears to be summed up of single photon responses with amplitudes smaller than about 50 pA. Their time course is similar to that of small single photon responses forming the so-called macroscopic C1 component in normal cells. The macroscopic current evoked by an intense flash has slow activation and deactivation kinetics and reaches a saturated amplitude of about 4-5 nanoamperes. The light-intensity dependence of the current evoked by flashes or by prolonged illumination has a slope of about 1 in log-log plots. The decay kinetics of the current is similar to that of the C1 component measured in normal cells after the block of the C2 component. Occasionally, the step-like current is superposed by large standard bumps. These bumps are blocked by the Ca2+-ATPase inhibitor cyclopiazonic acid, while the sustained inward current persists. We conclude that in unusual cells the light-activated current is identical to the C1 component of normal cells. The phospholipase C pathway that in normal cells presumably gives rise to the C2 component functions only with a low efficiency in unusual cells.

Animals↗