[Comparison of right thalamus and temporal cortex metabolite levels of drug-naive first-episode psychotic and chronic schizophrenia in patients]. / Ilaç Kullanmamis Ilk Psikotik Atak Hastalari ile Kronik Sizofreni Hastalarinda Sag Talamus ve Temporal Korteks Metabolit Seviyelerinin MRS ile Karsilastirilmasi.

OBJECTIVE: The aim of this study was to use Magnetic Resonance Spectroscopy (MRS) to investigate whether patients with chronic schizophrenia have different brain metabolite levels in the temporal cortex and thalamus than drug-naive first-episode patients. METHOD: We compared right-handed male first-episode patients (n=13) and chronic schizophrenic cases (n=15) with gender- and handedness-matched controls (n=10). Right temporal and right thalamic N-Acetylaspartate (NAA)/Creatine (Cre), NAA/Choline (Cho), and Cho/Cre ratios were obtained with MRS. RESULTS: Right temporal NAA/Cre, NAA/Cho, and right thalamus NAA/Cre ratios were significantly lower both in the chronic and first-episode patient groups when compared to normal controls (p<. 001), suggesting decreased neuronal integrity in both patient groups. There were no significant correlations between symptom severity and functional status with MRS variables (p=.027). These results suggested that both patient groups had neural integrity problems. Duration of illness (days) in the first-episode patients was significantly correlated with right temporal NAA/Cre and NAA/Cho. CONCLUSIONS: These results suggested that first-episode and chronic patients had significantly impaired neural integrity, particularly in the temporal cortex. It seems that in the acute phase of the first-episode, neural integrity impairment increased along with days elapsed without treatment.

Induction of glial glutamate transporters in adult mesenchymal stem cells.

Adult bone marrow mesenchymal stem cells are multipotent cells that can differentiate into a variety of mesodermal tissues. Recent studies have reported on their ability to also evolve into non-mesodermal cells, especially neural cells. While most of these studies revealed that manipulating these cells triggers the expression of typical neurone markers, less is known about the induction of neuronal- or glial-related physiological properties. The present study focused on the characterisation of glutamate transporters expression and activity in rat mesenchymal stem cells grown in culture conditions favouring their differentiation into astroglial cells. Ten days exposure of the cells to the culture supplement G5 was found to increase the expression of nestin (neuro-epithelial stem cell intermediate filament), an intermediate filament protein expressed by neural stem cells. Simultaneously, a robust induction of the high-affinity glutamate transporter GLT-1 (and GLAST) expression was detected by RT-PCR and immunocytochemistry. This expression was correlated with a highly significant increase in the Na+-dependent [3H]D-aspartate uptake. Finally, while glial fibrillary acidic protein immunoreactivity could not be detected, the induced expression of the astrocytic enzyme glutamine synthetase was demonstrated. These results indicate that in vitro differentiation of adult mesenchymal stem cells in neural precursors coincides with the induction of functional glutamate transport systems. Although the astrocytic nature of these cells remains to be confirmed, this observation gives support to the study of mesenchymal stem cells as a promising tool for the treatment of neurological diseases involving glutamate excitoxicity.

Aspartate transporter expression and activity in hypertrophic rat heart and ischaemia-reperfusion injury.

This study's rationale was that the expression and activity of aspartate transporters in hypertrophied hearts might be different from normal hearts, which could affect the use of aspartate in myocardial protection of hypertrophied hearts. mRNA expression of system X(ag)(-) transporters in hearts from normal (Wistar Kyoto) and hypertrophied (spontaneously hypertensive rat) rats was investigated by RT-PCR. EAAT3 protein expression in isolated cells and vesicles from normal and hypertrophied hearts was investigated by Western blotting. The same vesicles were also used to measure aspartate uptake. The effects of 0.5 mmol l(-1) aspartate supplementation on cardiac performance during ischaemia-reperfusion were investigated in isolated and perfused hearts. Both normal and hypertrophied hearts expressed EAAT1 and EAAT3 mRNA. EAAT3 protein expression was significantly greater in cells and vesicles from hypertrophied hearts compared to normal hearts. The velocity (V(max)) of aspartate uptake was faster at 24.4 +/- 2.2 pmol mg(-1) s(-1) in vesicles from hypertrophied hearts compared to 8.2 +/- 0.8 pmol mg(-1) s(-1) (P < 0.001, t test, n= 6, means +/-s.e.m.) in normal heart vesicles. The affinity (K(m)) was similar for both preparations. When recoveries were matched, 0.5 mmol l(-1) aspartate addition reduced reperfusion injury and increased functional recovery of hypertrophied hearts but not normal hearts. This was associated with a greater preservation of ATP, glutamate and glutamine and less lactate production during ischaemia in aspartate-treated hypertrophied hearts compared to all other experimental groups. These results suggest that increased aspartate transporter expression and activity in hypertrophy helps facilitate aspartate entry into hypertrophied cardiomyocytes, which in turn leads to improved myocardial protection.

LDL induced association of anionic liposomes with cells and delivery of contents as shown by the increase in potency of liposome dependent drugs.

PURPOSE: To establish whether anionic liposomes interact with the low-density lipoprotein (LDL) receptor, to determine the role of lipoproteins in this interaction, and whether the association causes functional delivery of encapsulated drugs. METHODS: The cell lines used were CV1-P and CHO wild type, both of which express the LDL receptor, and CHOldlA7, which lacks the LDL receptor. Cellular association of encapsulated methotrexate and fluorescein, labeled phosphatidylethanolamine in the lipid bilayer, was measured. Potency of three liposome dependent drugs (N-phosphonacetyl-L-aspartic acid, fluoroorotic acid, and methotrexate-gamma-aspartate) was also measured by growth inhibition. RESULTS: Association of liposomes containing at least 75 mol egg phosphatidylglycerol (ePG)/100 mol phospholipid with cells grown in defined medium supplemented with 1.0 mg/ml LDL was up to 30-fold higher with CV1-P or CHO wild type cells than with CHOldlA7, and 5-fold higher than association in defined medium lacking LDL. The addition of LDL did not yield any elevation of cellular association of distearoylphosphatidylglycerol liposomes. Increased association was paralleled by a corresponding increase in potency of all three liposome dependent drugs tested. CONCLUSIONS: ePG liposomes interact with the LDL receptor in an LDL-dependent fashion, and the interaction results in the delivery of contents to cells.

Bioavailability of US commercial magnesium preparations.

Magnesium deficiency is seen with some frequency in the outpatient setting and requires oral repletion or maintenance therapy. The purpose of this study was to measure the bioavailability of four commercially-available preparations of magnesium, and to test the claim that organic salts are more easily absorbed. Bioavailability was measured as the increment of urinary maginesium excretion in normal volunteers given approximately 21 mEq/day of the test preparations. Results indicated relatively poor bioavailability of magnesium oxide (fractional absorption 4 per cent) but significantly higher and equivalent bioavailability of magnesium chloride, magnesium lactate and magnesium aspartate. We conclude that there is relatively poor bioavailability of magnesium oxide, but greater and equivalent bioavailability of magnesium chloride, lactate, and aspartate. Inorganic magnesium salts, depending on the preparation, may have bioavailability equivalent to organic magnesium salts.

Therapeutic availability of iron administered orally as the ferrous gluconate together with magnesium-L-aspartate hydrochloride.

Since in vitro experiments had excluded interactions between Fe-gluconate (Fe-gluc) and magnesium-L-aspartate hydrochloride (MAH) in aqueous solutions the present in vivo studies seemed to be justified. Animal studies: Rats were kept on magnesium-(Mg)- and iron-(Fe)- sufficient and deficient diets. The intragastral administration of Fe-gluc significantly increased plasma Fe after 3 h, either given alone, or in combination with MAH (inducing hypermagnesemia). Same results were obtained when fortified diets were offered to Fe/Mg-deficient animals. Human studies: The combination of Fe-gluc (2 x 50 mg Fe per day, per os) plus MAH (2 x 7.5 mmol Mg per day, p.o.) was well tolerated by healthy volunteers. Single dose experiments revealed that Fe-gluc alone and in combination with MAH increased plasma Fe levels during 3 h to the same extent. Two groups of pregnant women with moderately reduced hemoglobin levels either received Fe-gluc (out-patients) or its combination with MAH (at least temporarily hospitalised because of preterm labor). Treatments were well tolerated. Hemoglobin levels did not further decrease, as expected without Fe supplements, during the course of pregnancy, thus indicating the therapeutic availability of the electrolytes in both study groups. Progesterone-induced constipation is frequently observed during pregnancy; hence stool softening reported by 50% of the women receiving Fe-gluc plus MAH (versus 33% in the Fe-gluc group) can be regarded as desirable effect. It is concluded that MAH does not interfere with the enteral absorption of Fe-gluc when both electrolytes are orally administered together. Taking both electrolytes together instead of 2 to 3 h apart from each other, as actually recommended, means a less complicated dosage regimen and probably improves compliance.

Different effects of three high-dose oral calcium salts on acid-base metabolism, plasma electrolytes and urine parameters of rats.

The oral calcium (Ca) load test has been applied to estimate the enteral absorbability of Ca salts in humans; provided that the deep bone compartments are filled up, excess Ca should be excreted in the urine. Using this "overflow model" three Ca salts were tested in rats at increasing oral doses of 0 to 14 mmol/kg body weight: CaCO3 and two other compounds containing chloride at a Ca:Cl ratio of 1:2 (CaCl2) and 1:1 (Ca-aspartate-hydrochloride). The carbonate was poorly absorbed and hence did not significantly affect acid-base metabolism nor urine pH. Both chloride-containing salts increased Ca excretion to a significantly higher degree in a dose-dependent manner; in contrast to the organic compound, the CaCl2 induced metabolic acidosis at 14 mmol/kg body weight. At decreasing base excess and urinary pH, renal excretion of Ca and of magnesium (Mg) increased, indicating that acid-base alterations must be considered when evaluating the oral load test. All Ca salts induced moderate hypomagnesemia pointing to decreased enteral absorbability of food-borne Mg in rats. Studies on volunteers reported in the literature suggest, however, that this effect is not relevant for humans.

The hypothalamo-cerebellar projection in the rat: origin and transmitter.

Hypothalamic neurons projecting to cerebellum were identified by retrograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the rat. Selective D-[3H]aspartate labelling was used to investigate whether any of these connections may use excitatory amino acids as transmitters. The WGA-HRP experiments revealed that the hypothalamo-cerebellar fibers have their main origins in the lateral, dorsal and posterior hypothalamic areas, and the tubero-mammillary nucleus, while smaller numbers of cells were observed in tuber cinereum, the anterior hypothalamic area, and the periventricular and paraventricular nuclei. After injections of D-[3H]aspartate into the cerebellar cortex, intense labelling of the olivocerebellar climbing fiber system was observed, but hypothalamic cells were not retrogradely labelled with this selective tracer. The absence of D-[3H]aspartate labelling indicates that hypothalamo-cerebellar neurons lack specific uptake mechanisms for excitatory amino acids, but it does not entirely preclude the possibility that some of these hypothalamic neurons may use such transmitters. Many cerebellar projecting cells were located in the tubero-mammillary nucleus, which is known to contain histaminergic and GABAergic neurons, and it was concluded that part of the hypothalamo-cerebellar pathways may use histamine and/or GABA as transmitters. The transmitter remains unknown for other parts of the hypothalamo-cerebellar pathways.

Retrograde transport of D-[3H]-aspartate injected into the monkey amygdaloid complex.

The possibility that certain of the afferents of the primate amygdaloid complex use an excitatory amino acid transmitter was evaluated by injecting D-[3H]-aspartate into the amygdala of two Macaca fascicularis monkeys. The distribution of D-[3H]-aspartate labeled neurons was compared with those labeled with the nonselective retrograde tracer WGA-HRP injected at the same location as the isotope. Retrogradely labeled cells of both types were observed in a variety of cortical and subcortical structures observed in a variety of cortical and subcortical structures and in discrete regions within the amygdala. D-[3H]-aspartate labeled neurons were observed in layers III and V of the frontal, cingulate, insular and temporal cortices. In the hippocampal formation, heavily labeled cells were observed in the CA1 region and in the deep layers of the entorhinal cortex. Of the subcortical afferents, the claustrum and the midbrain peripeduncular nucleus contained the greatest number of D-[3H]-aspartate labeled cells. Subcortical afferents that are not thought to use excitatory amino acids, such as the cholinergic neurons of the basal nucleus of Meynert, did not retrogradely transport the isotope. Within the amygdala, the most conspicuous labeling was in the paralaminar nucleus which forms the rostral and ventral limits of the amygdala. When the D-[3H]-aspartate injection involved the basal nucleus, many labeled cells were also observed in the lateral nucleus. Retrograde transport of D-[3H]-aspartate injected into the amygdala, therefore, appears to demonstrate a subpopulation of inputs that may use an excitatory amino acid transmitter.

Lack of effect of aspartame or of L-phenylalanine on photically induced myoclonus in the baboon, Papio papio.

The effects of large doses of L-phenylalanine and of aspartame on seizure susceptibility and severity have been assessed in baboons Papio papio from Senegal which show photosensitive epileptic responses similar to primary generalised epilepsy in man. L-Phenylalanine, 50, 150 or 450 mg/kg, or aspartame, 300 or 1000 mg/kg, were administered orally. Peak plasma L-phenylalanine concentrations of approximately 2000 mumoles/l occurred 1-4 h after the highest dose of L-phenylalanine or aspartame. The plasma L-phenylalanine to large neutral amino acid ratio increased approximately 30-fold at this time. Compared with water administration there were no changes in epileptic responses 1-5 h after either treatment. In this primate model of epilepsy acute increases in plasma phenylalanine concentration are neither pro- nor anticonvulsant.

Comparison of different caloric substrates on intestinal adaptation in the rat.

We compared the relative efficacy of medium- and long-chain triglycerides and dextrose on intestinal adaptation. Parenterally nourished rats received an isocaloric luminal infusion of one of these three substrates for 1 wk into either the jejunum or the ileum. Intestinal mass (mucosal weight and protein content) as well as the in vivo absorption of 5 mM glucose, valine, and aspartic acid were measured. In the jejunum, long-chain triglycerides were the most trophic, whereas in the ileum, long-chain triglycerides and dextrose were equally effective, but significantly more trophic than medium-chain triglycerides. In general, absorptive function was better maintained by dextrose and medium-chain triglycerides than long-chain triglycerides in the jejunum or by dextrose in the ileum. These data demonstrate that the jejunum and ileum respond differently to caloric substrates. Medium-chain triglycerides do not appear to have a clear superiority to long-chain triglycerides or dextrose in producing intestinal adaptation.

Glial uptake of excitatory amino acids influences neuronal survival in cultures of mouse hippocampus.

Several reports have described apparently normal survival and development of hippocampal and spinal cord culture preparations grown in Ham's F-12 medium, which contains 100 microM each of L-glutamate and L-aspartate. As at this concentration these amino acids are neurotoxic, some adaptive mechanism must occur to allow neuronal survival. We have investigated the mechanism underlying such adaptation. Dissociated cultures of mouse hippocampal neurons were grown in either Eagle's minimum essential medium or Ham's F-12 medium, supplemented with 5% horse serum. Analysis of neuronal density in cultures stained for neuron-specific enolase showed that although large numbers of neurons were present in mature cultures grown in either medium, neuronal survival in cultures grown continuously in F-12 was reduced to 41% compared to controls grown in Eagle's minimum essential medium. Physiological studies showed that those neurons which survived in F-12 did not lose their sensitivity to excitatory amino acids. In addition, the acute application of fresh, serum-free F-12 to 10-14-day-old cultures grown in either minimum essential medium or F-12 was highly neurotoxic. Three lines of evidence suggest that glial uptake of amino acids, and reduction of the extracellular concentration of glutamate and aspartate below neurotoxic levels, rather than receptor desensitization underlies the adaptive mechanism allowing neuronal survival. First, application of fresh F-12 produced large depolarizations, and profound neuronal swelling in cultures grown in F-12; however, after several hours swelling reversed suggesting a slow onset of the adaptive process. Second, pressure application of conditioned F-12 obtained from sister cultures also elicited depolarizations in neurons grown in F-12, but the amplitude of the underlying inward current was 25-30% of that produced by fresh F-12, suggesting a loss of potency of F-12 exposed to prolonged contact with hippocampal cultures. Third, measurement by high performance liquid chromatography showed reduction of aspartate concentrations to around 10% of those present in fresh F-12, within 24 h after exposing glial cell cultures to fresh F-12. It is concluded that cellular uptake mechanisms for amino acids have a strong impact on excitotoxicity in vitro, and most likely play an important role in protecting neurons from the potentially damaging action of high concentrations of excitatory transmitters in vivo. In addition our experiments help to explain the mechanisms permitting neuronal survival in cultures grown in Ham's F-12 medium, which when applied acutely to mature cultures is strikingly neurotoxic.

Oligodendroglial and astroglial heterogeneity in mouse primary central nervous system culture as demonstrated by differences in GABA and D-aspartate transport and immunocytochemistry.

Using simultaneous autoradiography and immunofluorescence we have investigated the functional heterogeneity amongst oligodendrocytes and astrocytes in primary mouse central nervous system (CNS) culture as expressed by differences in their ability to accumulate gamma-[3H]aminobutyric acid [( 3H]GABA) and D-[3H]aspartate. We have used a range of specific antibodies that identify oligodendrocytes and astrocytes, from precursor to fully mature cells, to address the question of whether all neuroglial cells are capable of expressing this function. Our results showing that A2B5-, 03-, and galactocerebroside-positive cells became heavily labelled with these two neuroactive amino acids, whereas cells expressing the myelin proteins 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and myelin basic protein (MBP) did not, demonstrate that this capacity is already present in oligodendrocytes at early developmental stages but may not extend to fully mature cells. Astrocytes in culture exhibited a large degree of variability with respect to their ability to transport GABA and D-aspartate. When grown in either serum-containing or serum-free hormone supplemented culture medium two morphologically distinct of glial fibrillary acidic protein (GFAP)-positive astrocyte were identified, process-bearing and epithelioid. Process-bearing cells became heavily labelled with the amino acids under both growth conditions, whereas, data showed that although epithelioid astrocytes were not, or only lightly, labelled with either amino acid in serum-containing cultures, when grown in serum-free culture medium they became more heavily labelled. Thus the expression, in culture, by epithelioid astrocytes, of one of the functions attributed to these cells is largely dependent on growth conditions.