Amelia Fiorilli

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.

Occurrence of Sialyltransferase Activity in the Synaptosomal Membranes Prepared from Calf Brain Cortex

The possible occurrence of sialyltransferase activity in the plasma membranes surrounding nerve endings (synaptosomal membranes) was studied, using calf brain cortex. The synaptosomal membranes were prepared by an improved procedure which provided: (a) a „nerve ending fraction” consisting of at least 85% well‐preserved nerve endings and containing only small quantities of membranes of intracellular origin; (b) a „synaptosomal membrane fraction” carrying high amounts of authentic plasma membrane markers (Na+‐K+ ATPase, 5′‐nucleotidase, sialidase, gangliosides) with values of specific activity four to fivefold higher than those in the „nerve ending fraction” and very small amounts of cerebroside sulphotransferase, marker of the Golgi apparatus, and of other markers of intracellular membranes (rotenone‐insensitive NADH and NADPH: cytochrome c reductases), the specific activities of which were, respectively, 0.5‐ and 0.7‐fold that in the „nerve ending fraction”. Thus the preparation of synaptosomal membranes used had the characteristics of plasma membranes and carried a negligible contamination of membranes of intracellular origin. The distribution of sialyltransferase activity in the main brain subcellular fractions (microsomes; P2 fraction; nerve ending fraction; mitochondria) resembled most closely that of thiamine pyrophosphatase, the enzyme known to be linked to the Golgi apparatus and the plasma membranes and of acetylcholine esterase, the enzyme known to be linked to either intracellular or plasma membranes. The enrichment of sialyltransferase activity in the „synaptosomal membrane fraction”, referred to the „nerve ending fraction”, was practically the same as that exhibited by authentic plasma membrane markers. All this is consistent with the hypothesis that in calf brain cortex sialyltransferase has two different subcellular locations: one at the level of intracellular structures, most likely the Golgi apparatus (as described by other authors), the other in the synaptosomal plasma membranes. The basic properties (pH optimum, V/S, V/t and V/protein relationships) and detergent requirements of the synaptosomal membrane‐bound sialyltransferase were established. The highest enzyme activities were recorded on exogenous acceptors, lactosylceramide and ds‐fetuin. The Km values for CMP‐NeuNAc were different using lactosylceramide and ds‐fetuin as acceptor substrates (0.57 and 0.135 mm, respectively); the thermal stability of the enzyme acting on glycolipid acceptor was higher than that on the glycoprotein acceptor; the effect of detergents was different when using glycoprotein from glycolipid acceptors; no competition was observed between lactosylceramide and ds‐fetuin. Thus the synaptosomal membranes carry at least two different sialyltransferase activities: one acting on lactosylceramide (and glycolipid acceptors), the other working on ds‐fetuin (and glycoprotein acceptors). Ganglioside GM3 was recognized as the product of synaptosomal membrane‐bound sialyltransferase activity working on lactosylceramide as acceptor substrate.

Casein-derived bioactive phosphopeptides: role of phosphorylation and primary structure in promoting calcium uptake by HT-29 tumor cells

Casein phosphopeptides β‐CN(1–25)4P and αs1‐CN(59–79)5P, from β‐ and αs1‐casein, respectively, both carrying the characteristic ‘acidic motif’ Ser(P)‐Ser(P)‐Ser(P)‐Glu‐Glu, were chemically synthesized and administered to HT‐29 cells differentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca2+]i due to influx of extracellular Ca2+. The response was quantitatively higher with β‐CN(1–25)4P than αs1‐CN(59–79)5P. The synthetic peptide corresponding to the ‘acidic motif’ was ineffective and the dephosphorylated form of β‐CN(1–25)4P almost inactive. The lack of the N‐terminally located five amino acids, or sequence modifications within the N‐terminal segment of β‐CN(1–25)4P, caused a total loss of activity, whereas the lack of the C‐terminal segment preserved activity. In conclusion, the influx of calcium into HT‐29 cells caused by β‐CN(1–25)4P appears to depend on the phosphorylated ‘acidic motif’ and the preceding N‐terminal region.

Caseinphosphopeptide-induced calcium uptake in human intestinal cell lines HT-29 and Caco2 is correlated to cellular differentiation.

Caseinphosphopeptides (CPPs) are considered as mineral carriers because of their ability to bind and solubilize calcium ions, with the possible role, yet to be definitely assessed, of improving calcium absorption at the intestinal level. Previous works demonstrated that CPPs improve calcium uptake, with increasing intracellular calcium concentration, by human differentiated tumor HT-29 cells, and that this effect correlates with the supramolecular structure of CPPs in the presence of calcium ions. The aim of the present study was to establish whether the CPP effect on calcium uptake is specific for HT-29 cells and depends on the differentiated state of the cells. To this purpose, HT-29 and Caco2 cells, two models of intestinal cells, were differentiated following appropriate protocols, including treatment with 1,25-(OH)2 vitamin D3. The CPP-dependent intracellular calcium rises were monitored at the single-cell level through fura2-fluorescence assays, and cell differentiation was assessed by biochemical and morphological methods. Results clearly showed that the ability to take up extracellular calcium ions under CPP stimulation is exhibited by both HT-29 and Caco2 cells, but only upon cell differentiation. This evidence adds novel support to the notion that CPPs favour calcium absorption, thus possibly acting as cellular bio-modulators and carrying a nutraceutical potential.

Interactions of ganglioside GM1 with human and fetal calf sera. Formation of ganglioside-serum albumin complexes

The interactions of ganglioside GM1 with human and fetal calf sera were studied, the following main results being obtained: (a) GM1, upon incubation with both sera gave origin to two GM1-protein complexes, which also occurred after interaction of GM1 with the albumin fractions prepared from the same sera. Instead no complex formation occurred using the albumin-free fractions. Therefore GM1 appeared to specifically bind serum albumin and to form GM1-albumin complexes. (b) GM1 binding to serum albumin started at ganglioside concentrations surely micellar (above 10(-6) M), was time and concentration dependent, and resulted in a relevant degree of GM1 complexation (up to 80% of total GM1 in human serum and up to 18% in fetal calf serum). (c) the binding kinetics appeared, in both serum and the correspondent albumin fraction, to be biphasic: in the first phase, occurring till about 2 . 10(-4) M GM1, the ratio between bound and total GM1 increased linearly with increasing GM1 concentration; in the second phase, occurring above 2 . 10(-4) M, the ratio remained practically constant. After these findings it should be expected that GM1, when present in serum containing systems, forms complexes with albumin. This should be approximately considered when studying the effects of exogeneous GM1 in in vivo and in vitro (tissue cultures) systems.

Casein phosphopeptide promotion of calcium uptake in HT‐29 cells − relationship between biological activity and supramolecular structure

Casein phosphopeptides (CPPs) form aggregated complexes with calcium phosphate and induce Ca2+ influx into HT‐29 cells that have been shown to be differentiated in culture. The relationship between the aggregation of CPPs assessed by laser light scattering and their biological effect was studied using the CPPs β‐CN(1–25)4P and αs1‐CN(59–79)5P, the commercial mixture CPP DMV, the ‘cluster sequence’ pentapeptide, typical of CPPs, and dephosphorylated β‐CN(1–25)4P, [β‐CN(1–25)0P]. The biological effect was found to be: (a) maximal with β‐CN(1–25)4P and null with the ‘cluster sequence’; (b) independent of the presence of inorganic phosphate; and (c) maximal at 4 mmol·L−1 Ca2+. The aggregation of CPP had the following features: (a) rapid occurrence; (b) maximal aggregation by β‐CN(1–25)4P with aggregates of 60 nm hydrodynamic radius; (c) need for the concomitant presence of Ca2+ and CPP for optimal aggregation; (d) lower aggregation in Ca2+‐free Krebs/Ringer/Hepes; (e) formation of bigger aggregates (150 nm radius) with β‐CN(1–25)0P. With both β‐CN(1–25)4P and CPP DMV, the maximum biological activity and degree of aggregation were reached at 4 mmol·L−1 Ca2+.

Exogenous gangliosides GD1b and GD1b-lactone, stably associated to rat brain P2 subcellular fraction, modulate differently the process of protein phosphorylation

GD1b and GD1b‐lactone (GD1b‐L) gangliosides bind to the same extent to a P2 crude membrane preparation from rat brain. After 30 min of incubation with 10−4, 105, and 10−6 Absolutions of ganglioside, 1,800, 450, and 100 pmol of ganglioside/mg of protein, respectively, were found to be stably associated to the P2 fraction. This association modifies the phosphorylation process of the P2 membrane proteins in a dose‐dependent manner, the maximal effect being reached at a ganglioside association of 1.85 nmol/mg of protein and in large part at 450 pmol/mg of protein. The effects of GD1b and GD1b‐L on the phosphorylation of five proteins, showing apparent molecular masses of 17, 20, 36, 41, and 44 kDa, were different after 0.5 min of phosphorylation reaction as well as after 15 min. After 0.5 min of reaction, in the presence of stably associated GD1b, the phosphorylation of the 36‐, 41‐, and 44‐kDa proteins was increased with reference to the control, whereas the phosphorylation of the 17‐ and 20‐kDa proteins was decreased. GD1b‐L exerted qualitatively similar effects only on the 44‐, 41‐, and 36‐kDa proteins and to a strongly reduced degree. After 15 min of reaction, only the phosphorylation of the 36‐kDa protein was stimulated by GD1b; GD1b‐L exerted a similar effect, but to a low degree.

Casein phosphopeptides promote calcium uptake and modulate the differentiation pathway in human primary osteoblast-like cells

Casein phosphopeptides (CPPs), originating by in vitro and/or in vivo casein digestion, are characterized by the ability to complex and solubilize calcium ions preventing their precipitation. Previous works demonstrated that CPPs improve calcium uptake by human differentiated intestinal tumor cell lines, are able to re-mineralize carious lesions in a dental enamel, and, as components of a diet, affect bone weight and calcium content in rats. The aim of the present study was to evaluate if CPPs can directly modulate bone cells activity and mineralization. Primary human osteoblast-like cells were established in culture from trabecular bone samples obtained from waste materials during orthopedic surgery. Commercial mixtures of bovine casein phosphopeptides were used. The CPP dependent intracellular calcium rises were monitored at the single cell level through fura2-fluorescence assays. Results show that CPPs: (i) stimulate calcium uptake by primary human osteoblast-like cells; (ii) increase the expression and activity of alkaline phosphatase, a marker of human osteoblast differentiation; (iii) affect the cell proliferation rate and the apoptotic level; (iv) enhance nodule formation by human SaOS-2. Taken together these results confirm the possibility that CPPs play a role as modulator of bone cell activity, probably sustained by their ability as calcium carriers. Although the exact mechanism by which CPPs act remains not completely clarified, they can be considered as potential anabolic factors for bone tissue engineering.

Uptake, cell penetration and metabolic processing of exogenously administered GM1 ganglioside in rat brain

GM1 ganglioside, after intravenous injection into rats, is absorbed and taken up by various organs and tissues, including brain. The capacity of brain to take up gangliosides, referred to weight unit, is comparable to that of kidney and muscle. After injection of [Gal-(3)H]GM1 a relevant portion of brain associated radioactivity resided in the soluble fraction and was of a volatile nature. After brain subcellular fractionation, the lysosomal, plasma membrane and Golgi apparatus fractions carried the highest specific radioactivity. In addition, an enriched fraction of brain capillaries was highly labelled, suggesting that GM1 ganglioside is also tightly bound to the vessel walls. The metabolic events encountered in brain by exogenous gangliosides were investigated, in detail, after intracisternal injection of [Sph-(3)H]GM1. The results obtained demonstrate that GM1 is extensively metabolized in brain. Besides the degradation products (GM2, GM3, lactosylceramide, glucosylceramide, ceramide), compounds of a biosynthetic origin were also found to be formed: these include GD1a, GD1b and sphingomyelin. All the above results could indicate that gangliosides, after intravenous administration to rats, are taken up by brain, bind to the capillary network, penetrate into neural cells, associate to both plasma membranes and intracellular structures and undergo metabolic processing with formation of a number of products of both catabolic and biosynthetic origin.

Human erythrocyte sialidase is linked to the plasma membrane by a glycosylphosphatidylinositol anchor and partly located on the outer surface.

Treatment of human erythrocyte ghosts with phosphatidylinositol-phospholipase C (PIPLC) fromBacillus cereus liberated the ghost-linked sialidase. Maximal release of sialidase (about 70% of total) was achieved by incubating ghosts at 37°C for 60 min, at pH 6.0, with PIPLC (PIPLC total units/ghost protein ratio, 4.5 each time) added at the beginning of incubation and every 15 min (four subsequent additions). Liberated sialidase was fully resistant to at least four cycles of rapid freezing and thawing and to storage at 4°C for at least 48 h. The liberated enzyme had an optimal activity at pH 4.2, degraded ganglioside GD1a better than methylumbelliferylN-acetylneuraminic acid (about fourfold), and gave aKm value of 2.56 · 10−4m and an apparentVmax of 2.22 mU per mg protein on GD1a. Treatment of intact erythrocytes with PIPLC (PIPLC total units/erythrocyte protein ratio, 8), under conditions where haemolysis was practically negligible, caused liberation of 10–12% of membrane linked sialidase, indicating that the enzyme is, at least in part, located on the outer surface of the erythrocyte membrane. It is concluded that the erythrocyte membrane sialidase is anchored by a glycosylphosphatidylinositol structure sensitive to PIPLC action, and is partly located on the outer surface.