EWHETA (Eat Well for a HEalthy Third Age) Project: novel foods to improve the nutrition in the elderly people.

Cardiovascular diseases (CVD) are the leading cause of mortality. However, by treating modifiable cardiovascular risk factors and following a healthy diet as the Mediterranean diet, we have opportunity to prevent CVD. In the EWHETA (Eat Well for a HEalthy Third Age) Project, our goal has been to develop novel foods ("Mediterranean Lasagne", MLs) in versions all nutritionally complete and well balanced in terms of calories, whole carbohydrates, fibers, source of vegetable proteins, and vegetable fats. MLs can be easy prepared at home (inexpensively) and used as fresh food or can be pre-prepared and used in residences for elderly people or in health care residencies. The project has saw the alliance between nutritionists and food and sensor scientists and the active involvement of older people in tasting the novel foods to achieve the final tasty versions of the MLs. We think that the nutritional components of these novel foods and its well-accepted taste, insert in a healthy diet and life style (fundamental aspects at every age), and could contribute to improve diet in the elderly people and prevent malnutrition.

Allozyme evidence supporting southwestern Europe as a secondary center of genetic diversity for the common bean.

Genetic diversity within a common bean ( Phaseolus vulgaris L.) collection, comprising 343 accessions from the Iberian Peninsula, was examined using six allozyme markers. Two major clusters corresponding to the Andean and Mesoamerican gene pools were identified. Both gene pools were characterized by specific alleles, with the former exhibiting Skdh(100), Me(100), Rbcs(100 or 98) and Diap-1(100), and the latter exhibiting Skdh(103), Me(100), Rbcs(100) and Diap-1(95). Some accessions from both clusters, deviating from these allozyme patterns, exhibited Skdh(100), Me(100), Rbcs(100) and Diap-1(95) or Skdh(103), Me(100), Rbcs(100) and Diap-1(100) allozyme profiles and were considered as putative hybrids.The levels of genetic variation has not been eroded since the introduction of the common bean from the American centers of domestication to the Iberian Peninsula. Instead, obvious signs of introgression between the two gene pools were observed, mainly among white-seeded genotypes. The intermediate forms adapted to the Iberian Peninsula could have emerged from initial recombination between Mesoamerican and Andean gene pools. The Iberian common bean germplasm is therefore more complex than previously thought, and contains additional diversity that remains to be explored for genetic and breeding purposes. The Iberian Peninsula could be considered as a secondary center of genetic diversity of the common bean, especially the large white-seeded genotypes.

Apoptosis in the dorsal lateral geniculate nucleus after monocular deprivation involves glutamate signaling, NO production, and PARP activation.

In mammals, visual experience during early postnatal life is critical for normal development of the visual system. Here we report that monocular deprivation for 2, 7, and 14 consecutive days causes p53 accumulation, cell death, and progressive loss of neurones in the dorsal lateral geniculate nucleus (dLGN) of newborn rats and these are prevented by NMDA and non-NMDA glutamate receptor antagonists, and by L-NAME, an inhibitor of nitric oxide synthesis. Monocular deprivation also increases dLGN levels of citrulline, the coproduct of nitric oxide synthesis, and this, as well as cell death and neuronal loss, is abolished by antagonists of glutamate receptors and by L-NAME. Finally, poly-(ADP-ribose) polymerase (PARP) knock-out mice appear to be protected from monocular deprivation-induced cell death. In conclusion, during early postnatal development of the rat visual system monocular deprivation causes excitotoxic, nitric oxide-mediated, cell death in the dLGN that appears to be apoptotic and also requires activation of PARP.

Systemic administration of cocaine, given alone or in combination with sensory stimuli, differentially affects L-arginine-nitric oxide metabolism in discrete regions of the brain of rat.

The effect of cocaine on brain regional metabolism of L-arginine to nitric oxide (NO) has been studied in rat by measuring the level of citrulline, the co-product of NO synthesis, using a HPLC based methodology. A single i.p. administration of 1 mg/kg cocaine, and a daily treatment for up to 5 consecutive days, failed to affect significantly citrulline content in the striatum, hippocampus and cortex. By contrast, in these regions of the brain a single or 5-day repeated higher dose of cocaine (10 mg/kg, i.p.) caused a significant increase in the co-product of NO synthesis and this has been abolished in a stereoselective fashion by L-NAME (10 mg/kg i.p. given 30 min before). Under cocaine high dose treatment, 1 h acoustic stimulation, which per se resulted ineffective, enhanced stimulant-induced increases in citrulline content seen in the striatum and abolished the increase of this amino acid observed in the hippocampus and cortex both after single or 5-day repeated injection of cocaine. In conclusion, these data demonstrate that cocaine stimulates the conversion of L-arginine to NO in the brain of rat and this is affected by concomitant exposure to acoustic stimulation.

Systemic administration of lithium chloride and tacrine but not kainic acid augments citrulline content of rat brain.

The effects of tacrine (5 mg/kg i.p.) in lithium chloride (LiCl; 12 mEq/kg i.p.)-pretreated (24 h beforehand) animals and of kainate (10 mg/kg i.p.) on brain citrulline, the co-product of nitric oxide (NO) synthesis, were studied in rats. High performance liquid chromatography analysis of whole brain tissue homogenates from rats treated with LiCl and tacrine revealed a significant increase in citrulline content before the onset of seizures. This effect was prevented in a stereoselective manner by N omega-nitro-L-arginine methyl ester (10 mg/kg i.p., given 20 min before tacrine), an inhibitor of NO synthase. By contrast, kainic acid (10 mg/kg i.p.) did not affect significantly brain citrulline during the pre-convulsive period. In conclusion, our data indicate that in rats seizures induced by LiCl and tacrine but not kainic acid are triggered by excessive NO production in the brain.

NMDA-dependent prostaglandin E2 release by human cultured astroglial cells is driven by nitric oxide.

The role of the L-arginine-NO pathway on the formation of PGE2 by human cultured astroglial cells incubated with NMDA has been investigated. Preincubation of T 67 astroglial cell line with NMDA (10-600 microM) produced a significant dose-dependent increase of both nitrite (the breakdown product of NO), PGE2 and cGMP levels in cell supernatant. This effect was inhibited by coincubation of cells with L-NAME (20-300 microM), an inhibitor of NO synthase showing that the release of PGE2 subsequent to NMDA receptor stimulation was driven by NO. The release of PGE2 but not elevation of nitrite and cGMP levels was affected by indomethacin (10 microM), an inhibitor of cyclooxygenase. The inhibitory effect of L-NAME on PGE2 release by NMDA-pretreated astroglial cells was reverted by arachidonic acid, showing that the effect of NO on PGE2 release occurred at the cyclo-oxygenase level. Thus, the present experiments demonstrate that the release of PGE2 by astroglial cells pretreated with NMDA is driven by activation of the L-arginine-NO pathway, and this may be relevant in the pathophysiological mechanisms where glutamatergic neurotransmission is involved.

Age-dependent changes of NO synthase activity in the rat brain.

We report that NO synthase activity, as expressed by citrulline and nitrite formation in brain homogenates, is decreased in 24-month old in comparison to 3-month old rats. In particular, a Ca(++)-dependent NO synthase activity was detected in homogenates obtained from cortical, hippocampal, cerebellar and lower brain stem slices from both 3- and 24 month-old rats. The amount of citrulline generated from L-arginine was significantly decreased in the hippocampus and lower brain stem by 40 and 48%, respectively. No changes were observed in NO synthase activity in cortical and cerebellar homogenates. Thus, the L-arginine-NO pathway seems to be impaired in selected areas of rat brain and this may contribute to the understanding of pathophysiological mechanisms underlying age-related cerebral disorders.

Lack of involvement of nitric oxide in the mechanisms of seizures and hippocampal damage produced by kainate and ouabain in rats.

The gross behavioural, electrocortical and neuropathological effects of kainate (10 mg/kg i.p,) and ouabain (1 micrograms, given into one dorsal hippocampus) were studied in rats. The effects of these treatments on nitric oxide synthase (NOS) activity in homogenates of hippocampus and cortex were also studied. Administration of kainate or ouabain produced motor and electrocortical seizures similar for latency to onset (approximately 15 min) and intensity (in all instances 80-100% of the treated rats showed behavioural and electrographic seizures). These effects were accompanied at 24 h by severe damage to all subsectors of the hippocampal formation and this concerned a similar proportion of the treated rats (n = 4-8 per treatment). No significant changes in nitric oxide synthase (NOS) activity were noted in the cerebral cortex and hippocampus of rats receiving injections of kainate and ouabain. In addition, pretreatment with N omega-Nitro-L-arginine methyl ester (300 micrograms, given into one lateral cerebral ventricle 15 min previously) was ineffective in preventing the effects of kainate and ouabain. In conclusion, present data suggest that excessive production of NO is not involved in the mechanisms triggering seizures and neurodegeneration produced by kainate or ouabain.

HIV coating gp 120 glycoprotein-dependent prostaglandin E2 release by human cultured astrocytoma cells is regulated by nitric oxide formation.

The role of the L-arginine-NO pathway on the formation of PGE2 by cultured astroglial cells incubated with the HIV coating glycoprotein gp120 was investigated. Preincubation of human cultured T 67 astrocytoma cells with gp 120 (100-500 nM) produced a significant increase of nitrite (the breakdown product of NO) and PGE2 in cell supernatants. The effect of gp 120 on both nitrite and PGE2 production was antagonized by inhibition of NO synthase by L-NAME (20-300 microM). The inhibition of gp120-induced PGE2 production by L-NAME was reverted by addition of arachidonic acid (30 microM), an effect antagonized by the cyclo-oxygenase inhibitor indomethacin (10 microM). Methylen bleu, an inhibitor of the biological activity of NO acting at the guanylate cyclase level failed to affect gp 120-mediated PGE2 release showing that the increase of cGMP subsequent to NO production was not involved in the modulatory activity of NO on arachidonic acid cascade. On the basis of present experiments we conclude that gp-120-induced release of PGE2 by astroglial cells is driven by NO, thereby contributing in the involvement of glial cells in HIV-related cerebral disorders.

Systemic administration of lithium chloride and tacrine increases nitric oxide synthase activity in the hippocampus of rats.

We planned to ascertain whether the administration of the anticholinesterase, tacrine (5 mg/kg i.p.), to rats pretreated 24 h before with lithium chloride (LiCl; 12 mEq/kg i.p.) produced any change in nitric oxide (NO) synthase activity in the hippocampus. A significant increase in hippocampal Ca(2+)-calmodulin-dependent NO synthase activity occurred 15 min after tacrine injection and was blocked by atropine (5 mg/kg i.p. given 15 min before tacrine) and by N omega-nitro-L-arginine methyl ester (300 micrograms given into one lateral cerebral ventricle 10 min before tacrine), a NO synthase inhibitor. A consistent cyclic guanosine 3',5'-monophosphate (cGMP) accumulation was also seen. In conclusion, the present results show that tacrine given to LiCl-pretreated rats produces a significant increase in NO synthase activity in the hippocampus and this may be responsible, at least in part, for seizures and related brain damage elicited by these drugs.

Cytokine-induced nitric oxide generation by cultured astrocytoma cells involves Ca(++)-calmodulin-independent NO-synthase.

The effect of several cytokines (IL1 beta and TNF alpha) on the inducible biosynthesis and release of NO by cultured astrocytoma cells was investigated and compared to that observed following pretreatment of cells with LPS. Preincubation for 4, 12, 24 and 48 h of astrocytoma cells with IL1 beta (10 ng ml-1), TNF alpha (500 U ml-1) and LPS (0.5 micrograms ml-1) enhanced their ability to inhibit thrombin-induced platelet aggregation through the release of an NO-like factor and increased nitrite levels in supernatants of stirred cells. The enhancement of NO production induced by cytokines as well as LPS was mediated by a time-dependent increase of NO-synthase activity in cell homogenates being mainly Ca(++)-calmodulin-independent. However, LPS activated earlier than cytokines the inducible NO-synthase and its effect was, at least in part, related to the release of IL1 beta by astrocytoma cells. In conclusion, the present data show, for the first time, that astrocytoma cells possess a cytokine-inducible Ca(++)-calmodulin-independent NO-synthase, whose activation seems to occur with a mechanism different from that described for LPS.

Neurotoxic effects induced by intracerebral and systemic injection of paraquat in rats.

1 The neurotoxic effects elicited by paraquat after systemic and intracerebral injection were studied in rats. 2 Intrahippocampal microinfusion of paraquat (0.1 mumol) produced behavioural stimulation and electrocortical (ECoG) excitation followed, at 24 h, by multifocal brain damage. Similarly, microinfusion of paraquat (0.2-0.4 mumol) into the locus coeruleus, substantia nigra or into the raphe nuclei, where noradrenergic, dopaminergic and serotonergic neurons are present, respectively, elicited potent excitotoxic effects (n = 6 rats per dose and area). A lower dose (0.01 mumol) of the herbicide or injection of the vehicle (1.0 microliter) did not produce any behavioural, ECoG or neurodegenerative effect. 3 After systemic administration, paraquat (20 mg kg-1 s.c.) evoked limbic motor seizures and ECoG epileptogenic discharges; in 10 out of 15 treated rats neuronal cell death was observed in the pyriform cortex, but not in other brain regions. A dose of 5 mg kg-1 was ineffective. 4 Among the regions of the brain studied, high concentrations of paraquat were detected in the pyriform cortex 24 h after systemic administration (5.0 and 20 mg kg-1 s.c.) lower levels being observed in the caudate nucleus. 5 In conclusion, paraquat, given systemically or intracerebrally in rats produces neurodegenerative effects.

Evidence that paraquat is able to cross the blood-brain barrier to a different extent in rats of various age.

The brain levels of paraquat were determined in rats of different age after systemic administration of several doses of the herbicide. Two week-, 3, 12 and 24 month-old rats were subcutaneously injected with paraquat (1, 2.5 and 5 mg/kg) and sacrificed 1 h later. A method based on ion-pair solid-phase extraction and reversed-phase high performance liquid chromatography was used to measure brain paraquat concentrations. The acute administration of paraquat resulted in dose-related and age-dependent brain concentrations of the herbicide, higher brain levels being detected in 12 and 24 month-old rats in comparison to young animals (3 month-old). In comparison with 3 month-old rats, higher concentrations of paraquat were also observed in the brain of 2 week-old animals. The present results demonstrate that in rats systemic treatment with paraquat gives rise to higher brain concentrations of this herbicide in very young and old rats in comparison with 3-month-old animals. This suggests that age-dependent changes in blood brain barrier permeability may account for these differences.