cDNA AFLP-based techniques for studying transcript profiles in horses.

The identification of differentially expressed genes is a fundamental prerequisite for understanding the molecular regulation of most physiological and pathological processes. Among the procedures employed to compare mRNA populations, those that are gel-based appear to hold great promise and are considered excellent tools for studying gene expression in species, such as the equine one, for which little genomic information is available. In the present study, we evaluated two techniques for studying mRNA profiles in horse tissue, one referred to the cDNA-amplified fragment length polymorphism (AFLP) that we called C-AFLP (classical cDNA-AFLP) protocol and the other to ordered differential display (ODD) with some modifications that we named S-AFLP (systematic cDNA-AFLP). Both techniques can be applied in live animals because of the small amount of sample required. We applied the S-AFLP to investigate horse transcript profile modifications during physical exercise. We found two transcripts that are mostly expressed during exercise and immediately after the end of it.

Effect of submaximal exercise on horse homocysteinaemia: possible implications for immune cells.

Physical exercise induces a reduction of immune defences and an imbalance of red-ox status. In this study plasma levels of cysteine and homocysteine (Hcy) were determined in horses before and after submaximal treadmill exercise as well as the effect on horse lymphocyte proliferation. The exercise induced a significant increase in plasma Hcy levels, which remained high both after the 20 min recovery period and after 2 h of rest. Moreover, a reduction of lymphocyte responsiveness to the proliferative stimulus induced by Concanavalin A was observed. The effects of different Hcy concentrations on the proliferative capacity of lymphocytes in culture were also tested. The results indicated that 10 microM of this amino acid can reduce the proliferative capacity of resting lymphocytes as well as their responsiveness to mitogen. Moreover, our results suggest that homocysteinaemia could be considered one of the parameters affected by physical exercise in horses and that this amino acid could be implicated in the effects of physical exercise on the immune system.

Effect of exercise training, selenium and vitamin E on some free radical scavengers in horses (Equus caballus).

Physical exercise increases both tissue needs for oxygen and cellular respiration and causes an overproduction of free radicals. When free radical generation exceeds the cell's antioxidant capacity tissue-damage develops due to oxidative stress. Therefore, it appears important to increase the scavenger ability of the tissues. Controlled training and dietary supplements may provide ways of doing this. As a model, we used 3-year-old racehorses (Equus caballus) which underwent a series of different physical exercise trials before and after 70 days of daily training and dietary supplements (vitamin E and selenium). The above treatments were able to increase both red blood cell resistance to the peroxidative stress induced in vitro and the glutathione peroxidase activity in lymphocytes. Moreover, they were also able to decrease malondialdehyde (MDA) concentration in the plasma as well as vitamin E consumption and the mobilisation of low molecular weight antioxidants (total peroxyl-radical trapping) following the physical exercise trials. The results obtained indicated that the training and diet supplements we used were able to significantly increase horse antioxidant defences in both the extracellular fluids and blood cells of our horses, thus decreasing peroxidative phenomena following physical exercise.

Physical exercise, oxidative stress and muscle damage in racehorses.

Since it has been suggested that lipid peroxidation following free radical overproduction may be one of the causes of physical exercise-induced myopathies and hemolysis in horses, we looked for the possible relationships between these phenomena and muscle fiber damage. We use a homogeneous group of Maremmana stallions which, after a 3-month training period, underwent a series of physical exercises of increasing intensity. We determined the contents of malondialdehyde (MDA), one of the main lipid peroxidation end-products, and glutathione the substrate of one of the most important free radical scavenger enzymes. We also measured creatine phosphokinase and serum lactate dehydrogenase isoenzyme activities whose modification may be indicative of muscle fiber damage. The results obtained indicated that the physical exercise we adopted was able to modify both MDA and glutathione contents in blood. However, its effect on some LDH isoenzyme activities suggested possible damage to tissues other than muscle.

Increased cerebrospinal fluid pyruvate levels in Alzheimer's disease.

Impaired energy metabolism is an early, predominant feature in Alzheimer's disease. In order to find out simple, reliable 'in vivo' markers for the clinical-biological typization of the disorder, we measured cerebrospinal fluid (CSF) glucose, lactate and pyruvate levels in patients suffering from dementia of Alzheimer type (DAT) and in healthy elderly controls. DAT group showed remarkably higher levels of pyruvate (P = 0.01), with no overlap with the values obtained in controls. CSF pyruvate levels were also significantly associated with the severity of dementia. Therefore, CSF pyruvate levels neatly separate DAT patients from controls, having also pathogenetic value.

Possible involvement of L-glycero-phosphoryl-ethanolamine in the phospholipid methylation pathway.

It is usually accepted that GPEtn is only an intermediate in glycerophospholipid degradation. However, some years ago J. P. Infante published a review-hypothesis concerning the possible involvement of GPEtn and GPCho in the biosynthesis of acyl-specific glycerophospholipids in mammalian tissues. This paper reports the results obtained by incubating brain cortex slices in the presence of labeled GPEtn. GPEtn enters the cells and the radioactivity of the precursor is found in both PE and PC. When these results were compared with those obtained by incubating brain slices in the presence of 3H-Etn, a significantly higher amount of radioactivity was found in PC, mainly after short incubations in the presence of low concentrations of the precursors. The slopes of the methylated forms of PE further underline the above differences found by comparing the results obtained with the two precursors. The results presented in this paper suggest a possible role of GPEtn in phospholipid biosynthesis and methylation pathway of PE.

Training-induced modifications in some biochemical defences against free radicals in equine erythrocytes.

Oxidative stress develops when the generation of free radicals exceeds the antioxidant capacity of cells or extracellular fluids. It can also occur as a result of physical exercise, and the pathogenesis of exercise-induced myopathies and haemolysis in horses may be related to changes in lipid peroxidation caused by free radicals. Cells have developed biochemical protection against oxidative stress and, as tissues seem to increase their antioxidant defences under chronic activation, training may be one of the ways of increasing antioxidant defences. Accordingly, we tested some enzymatic antioxidant activities as well as nonenzymatic antioxidants in horses undergoing special training. The results indicated a decrease in both chemical and biochemical defences against free radicals during training. It was deduced that the horses' diet may have been unable to provide the increased need for antioxidant defences resulting from training.

Acute phase reactant alpha 1-antichymotrypsin is increased in cerebrospinal fluid and serum of patients with probable Alzheimer disease.

Levels of alpha 1-antichymotrypsin (alpha 1-ACT) in cerebrospinal fluid (CSF) and serum from patients with probable Alzheimer disease (AD) of both early (e-AD) and late (l-AD) onset assessed by a competitive enzyme-linked immunosorbent assay were higher than those found in controls or in patients with vascular dementia (VD). A negative correlation between CSF levels of alpha 1-ACT and the stage of the disease was present in patients with both e-AD and l-AD. No difference in alpha 2-macroglobulin levels in CSF and serum from patients with e-AD, l-AD, VD, and nondemented controls was found. Serum concentrations of alpha 1-antitrypsin from l-AD subjects were within the normal range. Thus, increased levels of alpha 1-ACT in CSF and serum were specifically associated with AD, and the detection of this serpin in CSF may be useful in monitoring the progression of the disease.

Phospholipid base-exchange in brain microvessels.

The capillary endothelium of cerebral microvessels plays an important role in homeostasis within the central nervous system. The flux of fluids and solutes takes place through the lipid matrix of plasma membranes and the maintenance of their structural composition is necessary for cell membrane permeability and the cellular transport systems. Few studies have been carried out to clarify the relationships between brain microvessel lipid metabolism and the role of this metabolism. We have studied a particular aspect of the phospholipid metabolism, i.e. their hydrosoluble head group exchange with free choline, ethanolamine or serine, in capillaries from brains of both 4 month- old and 24 month-old rats. The results obtained indicate that microvessels possess the biochemical machinery of base-exchange reaction, whose activity rate appears unaffected by age.

Platelet MAO-B activity as a marker of behavioural characteristics in dementia disorders.

Both low and high platelet MAO-B (pMAO-B) activity is considered an indicator of increased vulnerability in psychopathology. How the activity of this peripheral enzyme can be linked with the sophisticated functions of the central nervous system (CNS) is not clear; in man, evidence exists that the genetic mechanisms determining the size or capacity of the central serotonin system are common to platelet and brain MAO. In the present study pMAO-B activity was evaluated in demented patients suffering from early-onset Alzheimer's disease (AD), late-onset Alzheimer's disease (SDAT), vascular dementia (VD), and controls. In these dementia categories, the relationship between pMAO-B activity and clinical features, and between pMAO-B activity and cerebrospinal fluid (CSF) monoamine metabolites (3-methoxy-4-hydroxyphenyl-glycol, MHPG; 5-hydroxy-indoleacetic acid, 5-HIAA; homovanillic acid, HVA) was also investigated. pMAO-B activity was significantly higher in SDAT patients, compared to controls and AD. Age, as covariate, failed to show any significant effect, and no association was found between pMAO-B activity and CSF monoamine metabolites. The correlation analysis between pMAO-B and neuropsychological scores showed a highly significant positive relationship with GBS-emotional impairment (N = 40, r = 0.72, p < 0.01) in the SDAT group. This result suggests the importance of platelet MAO-B activity as biological marker also in old-age dementias, namely senile dementia of Alzheimer type, where the increased activity of this enzyme might constitute a marker for vulnerability toward behavioural disturbance, i.e., emotional deterioration.

Linoleic acid passage through the blood-brain barrier and a possible effect of age.

It has already been established that the blood-brain barrier is readily crossed by unsaturated fatty acids, while saturated fatty acid transport appears to be protein mediated. When the passage of the fatty acids is tested in vivo by using perfusion buffers containing both linoleate and palmitate in different concentrations, linoleate is able to decrease the palmitate passage, while palmitate increases the linoleate passage. These results could be related to the effect of two fatty acids on the ratio between the fatty acids bound to the serum albumin and the free fatty acid pool, which is only available for transport through membranes. However, on the basis of some results obtained with aged rats, the possibility that a relationship may exist between palmitate and linoleate during their passage through the BBB is discussed. Moreover, it seems likely that in aged rats a moderate modification for fatty acids takes place in the BBB.

Defence mechanisms against free radical-induced damage in sheep, cattle and dog erythrocytes.

1. Erythrocytes from sheep were to be found the least resistant to peroxidative stress induced in vitro as compared to those from cattle and dogs. 2. The differences found among species in alpha-tocopherol content and glutathione peroxidase activity were probably insufficient to explain the lowest resistance. 3. One of the main reasons of the lowest resistance may be found in the membrane composition and characteristics (membrane fluidity).

Evidence for an altered dopamine beta-hydroxylase activity in migraine and tension-type headache.

Sympathetic dysfunction is often present in migraine. It has been suggested that serum dopamine-beta-hydroxylase (D beta H) can be taken as an index of peripheral sympathetic activity. We studied the serum D beta H activity in migraine with and without aura and in tension-type headache patients compared with healthy control subjects. The serum D beta H activity was significantly lower in migraine and tension-type headache patients than in the control group. No significant difference was observed among the three groups of patients studied. These findings suggest that patients with migraine and tension-type headache have a sympathetic hypofunction that may play an important role in the pathogenesis.

Arachidonic and palmitic acid utilization in aged rat brain areas.

We have previously demonstrated that the arachidonic acid (20:4) incorporation into brain lipids differs according to the age of the animals used and the experimental conditions adopted. These differences led to a further investigation of arachidonic acid uptake in both aged and adult rat brains, its transformation into CoA derivatives, its incorporation into diacyl-glycerols and polar lipids, and finally its oxidation to CO2. These metabolic parameters were then compared with those obtained after using the saturated fatty acid palmitate (16:0). In both cases slices or mitochondria from different brain areas of 24-month-old and 4-month-old rats were examined. The results obtained indicate that the uptake of the fatty acids into cells is not modified by age. However, the successive metabolic transformations of the acids are altered to a considerable extent. In particular, in 24-month-old animals (compared with 4-month-old rats) there is a significant decrease of 20:4 in its incorporation into lipids as well as its oxidation to CO2, while arachidonoyl-CoA content increases by about 50%. This increased amount of CoA derivative, which has a potent detergent effect, may interfere with membrane structure and affect membrane physiological functions. Furthermore, because the free arachidonate pool is maintained in a dynamic equilibrium with its esterified forms, the final result may be a perturbation of this equilibrium.

Linoleic acid metabolism in brain cortex of aged rats.

The linoleic acid metabolism was examined in the brain cortex of 4 month-old and 24 month-old rats. After the injection of [1-14C]-linoleate into the lateral ventricle of the brain the animals were sacrificed at 1,3 and 6 hours from the injection. The linoleate (18:2) incorporation into lipids, the presence of fatty acid peroxidation products, as well as the 18:2 transformation into elongated and desaturated derivatives were determined. Both an age-related reduction in linoleate incorporation rate into glycerophospholipids and a decrease in fatty acid turnover were found. Furthermore, in glycerophospholipids from 24 month-old rat brain cortex a higher level of hydroperoxide derivative of linoleate was found as compared to 4 month-old animals, and this damaged fatty acid is eliminated more slowly in aged rats than in adults. Finally, unlike 4 month-old animals, a stimulation of the transformation rate of linoleate into desaturation (6,9,12-C18:3) and elongation (8,11,14,C20:3) products was found in 24 month-old rat brain cortex. On the contrary, as far as arachidonic acid (one of the most important end products of the mechanism of linoleate modification) is concerned, the differences between aged and control animals were small, making it quite difficult to attribute a physiological meaning to this phenomenon.

Erythrocyte membrane composition in pregnancy-induced hypertension: evidence for an altered lipid profile.

OBJECTIVE: To investigate whether the increased membrane fluidity postulated as a possible contributing factor to the hypertensive states of pregnancy is related to the lipid composition of the erythrocyte membrane. DESIGN: An observational case control study. SUBJECTS: 30 women with pregnancy induced hypertension, 26 normotensive pregnant women matched for gestational age, and 10 normotensive non pregnant nulliparous women. INTERVENTIONS: Erythrocyte membranes were prepared from venous blood samples obtained from all the women. MAIN OUTCOMES MEASURES: Lipid analysis, including cholesterol to phospholipids ratio, distribution of phospholipid classes and fatty acid composition of total phospholipids in erythrocyte ghosts. RESULTS: The cholesterol/phospholipid ratio was significantly higher in the women with pregnancy induced hypertension compared with the normotensive pregnant women (mean 1.24, SD 0.31, 95% CI 1.12 to 1.35 vs mean 0.90, SD 0.09, 95% CI 0.86 to 0.94; P less than 0.01). Normotensive non-pregnant erythrocyte membrane cholesterol/phospholipid ratio was 0.88 (SD 0.11, 95% CI 0.79 to 0.96). The percentage distribution of different phospholipid classes and fatty acid composition was similar in all the four groups. CONCLUSIONS: The increased cholesterol/phospholipid ratio of the erythrocyte membrane found in pregnancy-induced hypertension represents one factor involved in the pathophysiology of this condition and a possible biochemical marker of the disease.

Platelet MAO-B activity and vitamin B12 in old age dementias.

Platelet MAO-B activity, serum vitamin B12 levels, and plasma folate were measured in patients suffering from presenile (AD) and senile (SDAT) dementia of Alzheimer-type, and vascular dementia (VD). MAO-B was higher in the SDAT group than in AD and controls. An inverse relationship between MAO-B activity and vit. B12 levels was documented in the whole group and in each category studied; furthermore, MAO-B was positively related to age. All the patients were then divided into two groups, according to vit. B12 levels (Group I: less than 200 pg/mL; Group II: greater than or equal to 200 pg/mL); Group I showed a significantly higher MAO-B activity with respect to Group II. The results indicate the existence of a negative association between platelet MAO-B activity and serum levels of vitamin B12 and confirm the existence of biological differences between presenile and senile dementia of Alzheimer type.

CSF monoamine metabolites in old age dementias.

Cerebrospinal Fluid (CSF) levels of the main metabolites of monoamines (MHPG, 5-HIAA, and HVA) were measured in patients with early onset (AD) and late-onset (SDAT) Alzheimer's disease, vascular dementia (VD), and elderly controls. Psychobehavioral assessment was carried out by means of MMSE and GBS. Mean MHPG levels did not differ from controls; 5-HIAA was lower in VD when compared to both controls and SDAT. HVA was decreased in AD, SDAT, and VD with respect to controls. Significant correlations between HVA and psycho-behavioral parameters were observed in SDAT and VD groups, whereas no relationship was documented in AD. The SDAT group was divided in SDAT-A (age at onset: greater than 65 less than or equal to 80 yr) and SDAT-B (age at onset: greater than 80 yr). SDAT-A had significantly lower CSF HVA values than SDAT-B (165 +/- 64 vs 235.7 +/- 85). SDAT-B HVA levels were similar to those observed in controls. Correlation analysis between HVA and neuropsychological variables was significant in SDAT-A, but not in SDAT-B. These results might support the evidence of SDAT heterogeneity.