Carlo Pieri

A gender--dependent genetic predisposition to produce high levels of IL-6 is detrimental for longevity.

Current literature indicates that elevated IL‐6 serum levels are associated with diseases, disability and mortality in the elderly. In this paper, we studied the IL‐6 promoter genetic variability at –174 C/G locus and its effect on IL‐6 serum levels in a total of 700 people from 60 to 110 years of age, including 323 centenarians. We found that the proportion of homozygotes for the G allele at –174 locus decreases in centenarian males, but not in centenarian females. Moreover, we found that, only among males, homozygotes for the G allele at –174 locus have higher IL‐6 serum levels in comparison with carriers of the C allele. On the whole, our data suggest that those individuals who are genetically predisposed to produce high levels of IL‐6 during aging, i.e. –174 locus GG homozygous men, are disadvantaged for longevity.

Serum paraoxonase is reduced in type 1 diabetic patients compared to non-diabetic, first degree relatives; influence on the ability of HDL to protect LDL from oxidation

Paraoxonase is a serum enzyme with an anti-oxidant function, protecting low density lipoproteins (LDL) from oxidative modifications. Diabetic patients are suggested to be at greater risk of oxidative stress, which may contribute to the significantly higher incidence of vascular disease in this population. Less efficient protection mechanisms may be one feature of the greater susceptibility to oxidation in diabetes. In this context, the present study examined the hypothesis that serum paraoxonase is reduced in type 1 (insulin-dependent) diabetic patients and that the reduction can affect the anti-oxidant capacity of HDL. Serum paraoxonase concentrations and activities were compared in type 1 patients and first degree, non-diabetic relatives with particular attention paid to the confounding effects of paraoxonase gene polymorphisms. In addition, the ability of HDL-paraoxonase to protect low density lipoproteins from oxidation was analysed in an in vitro system. Serum concentrations and enzyme activities of paraoxonase were significantly lower in type 1 patients compared to non-diabetic, first degree relatives. The differences were independent of promoter and coding region polymorphisms, which influence serum concentrations and activities of the enzyme. Overall, paraoxonase concentrations were a mean 13.3+/-4.5% lower (P<0.02) in type 1 patients. Specific activities did not differ between diabetic and non-diabetic groups. The concentration ratios of LDL cholesterol:paraoxonase (1.37+/-0.51 vs. 1.18+/-0.37, P=0.003) and apolipoprotein B:paraoxonase (0.84+/-0.33 vs. 0.71+/-0.40; P=0.012) were significantly higher in diabetic patients, consistent with a reduced capacity to protect LDL from oxidation. In vitro oxidation studies showed that a significantly higher level of lipid hydroperoxides was generated in LDL in the presence of HDL, containing paraoxonase levels equivalent to those of type 1 patients, compared to HDL containing paraoxonase levels equivalent to those of control subjects (mean difference 8.1%, P<0.05). The study demonstrates that serum concentrations of the antioxidant enzyme paraoxonase are significantly lower in type 1 (insulin-dependent) diabetic patients compared to non-diabetic, first-degree relatives, independently of known gene polymorphisms. Concentrations are reduced to an extent that can affect its anti-oxidant capacity. The results are consistent with the contention that modifications to serum paraoxonase in type 1 patients can increase risk of lipoprotein oxidation and, consequently, risk of vascular disease.

Genetic analysis of Paraoxonase (PON1) locus reveals an increased frequency of Arg192 allele in centenarians

Human Paraoxonase (PON1) is a High-Density Lipoprotein (HDL)-associated esterase that hydrolyses lipo-peroxides. PON1 has recently attracted attention as a protective factor against oxidative modification of LDL and may therefore play an important role in the prevention of the atherosclerotic process. Two polymorphisms have been extensively studied: a Leucine (L allele) to Methionine (M allele) substitution at codon 55, and a Glutamine (A allele) to Arginine (B allele) substitution at codon 192. We have examined these two aminoacidic changes in 579 people aged 20 to 65 years old, and 308 centenarians. We found that the percentage of carriers of the B allele at codon 192 (B+ individuals) is higher in centenarians than in controls (0.539 vs 0.447), moreover we found that among the B+ individuals, the phenomenon was due to an increase of people carrying M alleles at codon 55 locus. In conclusion, we propose that genetic variability at PON1 locus affects survival at extreme advanced age.

Mitochondrial dysfunctions during aging: vitamin E deficiency or caloric restriction--two different ways of modulating stress.

Caloric restriction (CR), which has been demonstrated to offset the age-associated accrual of oxidative injury, involves a reduction in calory intake while maintaining adequate nutrition, preserves the activities of antioxidant enzymes in postmitotic tissues, maintains organ function, opposes the development of spontaneous diseases, and prolongs maximum life span in laboratory rodents. It has been proposed that reductions in Reactive Oxygen Species (ROS) production and cellular oxidative injury are central to the positive effects of CR. In the present investigation we studied the effect of CR and of a vitamin E deprived diet on mitochondrial structure and features in the liver of rats during aging, in order to ascertain the extent of modifications induced by these experimental conditions. CR rats displayed structural and functional mitochondrial properties (fatty acid pattern, respiratory chain activities, antioxidant levels, and hydroperoxide contents) similar to those of younger rats whilst vitamin E deficient rats appeared older than their own age. The mitochondria of the former, together with those of young rats, possessed the lowest Coenzyme Q9, hydroperoxide, and cytochrome contents as well as a suitable fatty acid membrane composition. Our study confirms that CR is a valuable tool in limiting aging-related free-radical damage also at mitochondrial liver level.

Food restriction in female Wistar rats: V. Lipid peroxidation and antioxidant enzymes in the liver

The activities of antioxidant enzymes as well as the levels of basal and enzyme induced peroxidation have been investigated in liver of female Wistar undernourished rats. Food restriction was applied starting from the age of 3.5 months by feeding the animals on every-other-day schedule (EOD). Diet restriction prevented the age-dependent increase of basal and enzyme induced lipid peroxidation in both mitochondrial and microsomal liver membrane preparations. The activities of antioxidant enzyme, i.e. superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) of liver decreased during aging in ad libitum fed rats. In the diet conditioned animals, a small increase of SOD and a complete recovery of CAT activities were observed. Present data support that food restriction improved the protection against peroxidation, and this may be in close relationship with the life prolonging effect of such a treatment.

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.

Studies on the life prolonging effect of food restriction: glutathione levels and glyoxalase enzymes in rat liver

Cytosolic and mitochondrial levels of glutathione (GSH) as well as the activities of glyoxalase I (GI) and glyoxalase II (GII), GSH-dependent enzymes involved in the detoxification of 2-ketoaldehydes, were investigated in the liver of ad libitum (AL) fed and food restricted (FR) rat during aging. Both cytosolic and mitochondrial GSH level was lower in old than in adult AL fed rats. Food restriction did not prevent this decrease, but its extent was attenuated considering the cytosolic GSH. As regards the mitochondrial GSH, its content was higher in adult FR animals than in the age-matched AL fed ones. Thus, the subsequent age-dependent decrease of GSH, occurring also in FR animals, resulted in a thiol concentration not different from that observed in young and adult AL fed animals. Considering the enzymatic activities, cytosolic GI decreased in old rats irrespective of diet, whereas GII activity remained constant in all the experimental groups. The higher glutathione content found in both cellular compartments of old FR rats as compared to the old AL fed ones, could help to explain the life prolonging effect of FR treatment. Moreover, the observation that the activity of glyoxalases was not influenced by food restriction does not necessarily mean that the cells of diet-conditioned animals are scarcely protected against the toxic effect of methylglyoxal. Indeed, the production of this compound should be lower in FR animals as compared to AL fed ones, due to the lower level serum glucose concentration during the life span of the former with respect to the latter group.

Melatonin regulates the respiratory burst of human neutrophils and their depolarization

Pieri C, Recchioni R, Moroni F, Marcheselli F, Marra M, Marinoni Silvia, Di Primio R. Melatonin regulates the respiratory burst of human neutrophils and their depolarization. J. Pineal Res. 1998; 24:43–49.

Two-dimensional receptor patterns in the plasma membrane of cells. A critical evaluation of their identification, origin and information content

A concise review is presented on the nature, possible origin and functional significance of cell surface receptor patterns in the plasma membrane of lymphoid cells. A special emphasize has been laid on the available methodological approaches, their individual virtues and sources of errors. Fluorescence energy transfer is one of the oldest available means for studying non-randomized co-distribution patterns of cell surface receptors. A detailed and critical description is given on the generation of two-dimensional cell surface receptor patterns based on pair-wise energy transfer measurements. A second hierarchical-level of receptor clusters have been described by electron and scanning force microscopies after immuno-gold-labeling of distinct receptor kinds. The origin of these receptor islands at a nanometer scale and island groups at a higher hierarchical (mum) level, has been explained mostly by detergent insoluble glycolipid-enriched complexes known as rafts, or detergent insoluble glycolipids (DIGs). These rafts are the most-likely organizational forces behind at least some kind of receptor clustering [K. Simons et al., Nature 387 (1997) 569]. These models, which have great significance in trans-membrane signaling and intra-membrane and intracellular trafficking, are accentuating the necessity to revisit the Singer-Nicolson fluid mosaic membrane model and substitute the free protein diffusion with a restricted diffusion concept [S.J. Singer et al., Science 175 (1972) 720].

Ligand and voltage gated sodium channels may regulate electrogenic pump activity in human, mouse and rat lymphocytes

Bretylium tosylate - a sodium channel opener - resulted in an increase of membrane potential of depolarized human, rat and mouse T and B lymphocytes. Flow cytometric membrane potential measurements with bis-oxonol revealed that the above hyperpolarizing effect was amiloride, ouabain, tetrodotoxin, azide and temperature sensitive. The effect showed an absolute dependence on the extracellular sodium but it was insensitive to the extracellular Ca2+ level. The voltage gating of the effect can be eliminated by either an increase of the extracellular potassium concentration or low doses of veratrin. The existence of a voltage and ligand gated sodium channel is suggested in the plasma membrane of all kinds of lymphocytes. The hyperpolarization is explained by an increased activity of the electrogenic sodium-potassium ATP-ase. Induced opening of such sodium channels may regulate the electrogenic pump activity and indirectly cell activation.