Benvenuto Cestaro

Age-Related Differences in Synaptosomal Peroxidative Damage and Membrane Properties

Abstract: Young, adult, and old rats were used to study the effect of age on the integrity and functioning of brain synaptosomes. An evaluation was made of the differences in lipid composition, membrane fluidity, Na+,K+‐ATPase activity, and susceptibility to in vitro lipid peroxidation. There was an age‐related increase in synaptosomal free fatty acids, with no modification in acyl chain composition, and a decrease in membrane phospholipids which increased the cholesterol/phospholipid mole ratio. With altered lipid composition, there was a corresponding age‐dependent decrease in membrane fluidity, a reduction of Na+,K+‐ATPase activity, and an overall greater susceptibility to in vitro lipid peroxidation. Furthermore, lipid peroxidation promoted strong modifications of the membrane fluidity, lipid composition, and Na+,K+‐ATPase activity just as aging did, thus indicating a possible contribution of oxidative damage to aging processes. The cases studied revealed that the greater responsiveness of old membranes to in vitro lipid peroxidation resulted in the highest degree of membrane alteration, indicating that all pathological states known to promote a peroxidative injury can have even more dramatic consequences when they take place in old brain.

Biochemical assessments of oxidative stress, erythrocyte membrane fluidity and antioxidant status in professional soccer players and sedentary controls

Background  Physical exercise is characterized by an increase in oxygen consumption by the whole body. This leads to a decrease in antioxidant levels that could promote both an increase in the markers of lipoprotein peroxidation and damage to the erythrocyte membrane with consequent modification of membrane fluidity.

Studies on the antioxidant activity of milk caseins

The antioxidant properties of milk casein subunits (alpha-casein, beta-casein and kappa-casein) were evaluated in liposomal models. All the subunits of casein are able to inhibit Fe-induced peroxidation of arachidonic acid inserted into multilamellar liposomes of dipalmitoylphosphatidylcholine (0.2 mM and 0.8 mM, respectively). The peroxidation was monitored as thiobarbituric acid reactive substances, and the strongest inhibitory effect occurred when 500 micrograms of alpha-casein were added to 0.5 ml of liposomal suspension. At this concentration, peroxidation was completely inhibited in our experimental conditions (incubation for 2 h at room temperature, with a mixture of ferrous sulfate and ascorbate, 50 and 500 microM final concentration, respectively). The mechanisms of antioxidant action are complex, but the strongest effect is achieved by modifying the Fe2+/Fe3+ equilibrium; in fact, caseins seem to favour the autoxidation of iron, and thus inhibit lipid peroxidation.

Electron paramagnetic resonance studies on the fluidity and surface dynamics of egg phosphatidylcholine vesicles containing gangliosides

The influence of different gangliosides (GM1, GD1a, GT1b) on the fluidity and surface dynamics of phosphatidylcholine small unilamellar vesicles was studied by electron paramagnetic resonance. 5- and 16-nitroxystearic acid, sounding respectively the region close to the surface and that close to the hydrophobic core of the vesicle, were employed as spin-label probes. The signals released by 5-nitroxystearic acid showed that the presence of gangliosides reduced the mobility of the hydrocarbon chains around the probe. The effect increased by increasing ganglioside concentration, and diminished from GM1 to GD1a and GT1b. The decrease of membrane fluidity was also monitored by the 16-nitroxystearic acid probe. On addition of Ca2+ the fluidity of ganglioside-containing vesicles (as signalled by the 5-nitroxystearic acid probe) promptly decreased, therefore returning slowly to the original value. It is suggested that gangliosides cause strong side-side head group interactions on the bilayer surface--between ganglioside oligosaccharide chains and between ganglioside and phosphatidylcholine polar portions--which lead the lipid chains to assembly in a more rigid fashion. The influence of Ca2+ is interpreted as due to lateral phase separation in the vesicle membrane. This phenomenon can be related to the formation or stabilization of ganglioside clusters on the vesicle surface.

Studies on peroxidation of arachidonic acid in different liposomes below and above phase transition temperature

The mechanism of Fe-induced peroxidation of arachidonic acid (AA) in small unilamellar vesicles (SUV) of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE) was studied below and above gel to liquid-crystalline phase transition temperature (Tm). In both liposomes the AA peroxidation resulted higher in the temperature range below Tm, but the extent of malonyldialdehyde (MDA) formation was dramatically lower in DPPE vesicles when compared with corresponding DPPC liposomes. A possible explanation for this is discussed.

Studies on brain cytosol neuraminidase: I. Isolation and partial characterization of two forms of the enzyme from pig brain

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.

Studies on peroxidation processes of model membranes and synaptosomes: role of phosphatidic acid

Dipalmitoylphosphatidic acid (DPPA) was found to exert a strong inhibitory effect on Fe-induced peroxidation of arachidonic acid inserted into liposomal dipalmitoylphosphatidylcholine (DPPC) vesicles. This inhibition was quite effective both below and above the phase transition temperature of the liposomes. Moreover, we demonstrated the antiperoxidative activity of phosphatidic acid (PA) in synaptosomal membranes. PA enriched synaptosomes were prepared by the stimulation of the endogenous phospholipase D activity or by the incubation of the synaptosomes with Streptomyces chromofuscus phospholipase D. The possible contribution of PA to the in vivo defense mechanism against free radical-induced damage is discussed.

Anti-oxidant, anti-glycant, and inhibitory activity against α-amylase and α-glucosidase of selected spices and culinary herbs

Aqueous and methanol extracts of dry sage, rosemary, basil, parsley, chili, garlic and onion were analyzed to investigate their anti-oxidant and anti-glycant activities and in vitro inhibitory potential against enzymes involved in glycemic regulation. The aqueous extracts of rosemary and sage were the richest in phenolic compounds and showed the highest ability in binding iron and inhibiting DPPH, superoxide radicals and advanced glycation end-product production, lipid peroxidation, and the activity of α-glucosidase and α-amylase. On the other hand, the methanol extracts of both these Labiatae were less efficient than those of garlic, onion, parsley and chili in scavenging hydroxyl radicals. As far as protein glycation is concerned, methanol extracts were more effective in inhibiting the production of Amadori compounds and the aqueous ones in preventing advanced glycation end-product formation. Therefore these spices may be preventive not only against cardiovascular diseases but also type 2 diabetes.

Effects of arginine, S-adenosylmethionine and polyamines on nerve regeneration

Introduction ‐ Axon growth and axon regeneration are complex processes requiring an adequate supply of certain metabolic precursors and nutrients. Material and methods ‐ This article reviews the studies examining some of the processes of protein modification fundamental to both nerve regeneration and to the continuous and adequate supply of specific factors such as arginine, S‐adenosylmethionine and polyamines. Results ‐ The process of arginylation notably increases following nerve injury and during subsequent regeneration of the nerve, with the most likelyfunction of arginine‐modification of nerve proteins being the degradation of proteins damaged through injury. It appears that defective methyl group metabolism may be one of the leading causes of demyelination, as suggested by the observation of reduced cerebrospinal fluid concentrations of s‐adenosylmethionine (SAMe) and 5‐methyltetrahydrofolate, the key metabolites in methylation processes, in patients with a reduction in myelination of corticospinal tracts. Polyamine synthesis, which depends strongly on the availability of both SAMe and arginine, markedly increases in neurons soon after an injury. This “polyamine‐response” has been found to be essential for the survival ofthe parent neurons after injury to their axons. Polyamines probably exert their effects through involvement in DNA, RNA and protein synthesis, or through post‐translational modifications that areindicated as the most relevant events of the “axon reaction.” Conclusions ‐ Nerve regeneration requires the presence of arginine, s‐adenosylmethionine, and polyamines. Further studies are needed to explore the mechanisms involved in these processes.

N-Pyrene dodecanoyl sulfatide as membrane probe: a study of glycolipid dynamic behavior in model membranes

An N-linked pyrene-dodecanoyl sulfatide was employed to measure the ratio of excimer fluorescence to monomer fluorescence intensities (E/M). The E/M values provided information about both the dynamic behavior and the structural distribution of the labelled glycolipid in note dispersion of micellar sulfatides and multilamellar vesicles of different phospholipids. Most of the labelled sulfatide seems to be located in domains sequestered from the surrounding phospholipids still above the phase transition temperature of the vesicles. The glycolipids sequestered in these domain environments are less sensitive to the structural changes that the addition of cholesterol or Ca2+ can induce in the phospholipid regions during the phase transition.