N-Methyl-D-Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo

There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.     

Amino Acid Derivatives as Palmitoylethanolamide Prodrugs: Synthesis,In Vitro Metabolism and In Vivo Plasma Profile in Rats

Palmitoylethanolamide (PEA) has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, we prepared carbonates, esters and carbamates at the hydroxyl group of PEA. Chemical stability (pH 7.4) and stability in rat plasma and liver homogenate were evaluated by in vitro assays. Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. Ester derivatives, prepared by conjugating PEA with various amino acids, allowed to modulate the kinetics of PEA release in plasma and stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but provided lower plasma concentrations than those obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization.     

Influence of Amino Acids Shiff Bases on Irradiated DNA Stability In Vivo

To reveal protective role of the new Mn(II) complexes with Nicotinyl-l-Tyrosinate and Nicotinyl-l-Tryptophanate Schiff Bases against ionizing radiation. The DNA of the rats liver was isolated on 7, 14, and 30 days after X-ray irradiation. The differences between the DNA of irradiated rats and rats pre-treated with Mn(II) complexes were studied using the melting, microcalorimetry, and electrophoresis methods. The melting parameters and the melting enthalpy of rats livers DNA were changed after the X-ray irradiation: melting temperature and melting enthalpy were decreased and melting interval was increased. These results can be explained by destruction of DNA molecules. It was shown that pre-treatment of rats with Mn(II) complexes approximates the melting parameters to norm. Agarose gel electrophoresis data confirmed the results of melting studies. The separate DNA fragments were revealed in DNA samples isolated from irradiated animals. The DNA isolated from animals pre-treated with the Mn(II) chelates had better electrophoretic characteristics, which correspond to healthy DNA. Pre-treatment of the irradiated rats with Mn(II)(Nicotinil-l-Tyrosinate) and Mn(II)(Nicotinil-l-Tryptophanate)₂ improves the DNA characteristics.     

Derivatives of Aminoquinones with N-protected Amino Acids

The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives. N,N'-Diisopropylcarbodiimide/1-hydroxybenzotriazole or N,N'-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Derivatives of Aminoquinones with N-protected Amino Acids

Summary The synthesis of derivatives of aminoquinones with N-protected amino acids is reported here. 2-Amino-1,4-benzoquinone and 2-amino-1,4-naphthoquinone, prepared by the azide method in yields of 60 and 95% respectively, were coupled with N-Boc-protected amino acids including glycine, serine, proline and tyrosine, to give the correspondening derivatives.N, N′-Diisopropylcarbodiimide/1-hydroxybenzotriazole orN, N′-dicyclohexylcarbodiimide/HOBt used as coupling reagents provided the expected products in satisfactory yields and purities as supported by TLC, HPLC and spectral analysis.     

Brain Amino Acids During Hyponatremia In Vivo: Clinical Observations and Experimental Studies

Hyponatremia is a highly morbid condition, present in a wide range of human pathologies, that exposes patients to encephalopathic complication and the risk of permanent brain damage and death. Treating hyponatremia has proved to be difficult and still awaits safe management, avoiding the morbid sequelae of demyelinizing and necrotic lesions associated with the use of hypertonic solutions. During acute and chronic hyponatremia in vivo, the brain extrudes the excessive water by decreasing its content of electrolytes and organic osmolytes. At the cellular level, a similar response occurs upon cell swelling. Among the organic osmolytes involved in both responses, free amino acids play a prominent role because of the large intracellular pools often found in nerve cells. An overview of the changes in brain amino acid content during hyponatremia in vivo is presented and the contribution of these changes to the adaptive cell responses involved in volume regulation discussed. Additionally, new data are provided concerning changes in amino acid levels in different regions of the central nervous system after chronic hyponatremia. Results favor the role of taurine, glutamine, glutamate, and aspartate as the main amino acid osmolytes involved in the brain adaptive response to hyponatremia in vivo. Deeper knowledge of the adaptive overall and cellular brain mechanisms activated during hyponatremia would lead to the design of safer therapies for the hyponatremic patient.     

Solubilities Studies of Basic Amino Acids

The solbilities of l-basic amino acids in the type of free, monohydro-chloride and dihydrochloride in water were determined, and the results were formulated as follows.

l-Arginine: log S = 0.9770+0.01345t (t is from 0°~70°C)

l-Histidine: log S = 0.3627+0.00905t (t is from 0°~70°C)

l-Arginine monohydrochloride: log S = 1.6532+0.01301t (t is from 0°~70°C)

l-Lysine monohydrochloride dihydrate: log S = 1.6990+0.01294t (t is from 0°~55°C)

l-Lysine monohydrochloride monohydrate: log S = 1.7404+0.01256t (t is from 55°~70°C)

l-Lysine dihydrochloride: log S = 2.2138+0.00409t (t is from 0°~70°C)

l-Histidine dihydrochloride: log S = 1.9085+0.00265t (t is from 0°~70°C)

Three component systems (basic amino acid, hydrochloric acid, water) were studied and the solubilities in mixed solution system of alcohol-water were also investigated.     

Synthesis of New Pseudonucleosides Containing Sulfamylated Derivatives of Natural Amino Acids as Aglycone

Abstract

New chiral sulfahydantoins have been synthesized via alkaline cyclisation, starting from symetric sulfamide derivatives of natural amino acids. Tetraacetyl ribofuranose was used in the glycosylation step in order to obtain the pseudonucleosides in β-anomeric configuration.     

Purification and Characterization of the Crown Gall-specific Enzyme, Octopine Synthase

A single enzyme catalyzes the synthesis of all four N²-(1-carboxyethyl)-amino acid derivatives found in a crown gall tumor tissue induced by Agrobacterium tumefaciens (E. F. Sm. and Town.) Conn strain B6 on sunflower (Helianthus annuus L.). This enzyme, octopine synthase, has been purified by ammonium sulfate fractionation and chromatography on diethylaminoethylcellulose, blue agarose, and hydroxylapatite. The purified enzyme has all the N²-(1-carboxyethyl)-amino acid synthesizing activities found in crude preparations, and the relative activities with six amino acids remain nearly constant during purification. Although the maximum velocities (V) and Michaelis constants (K_m) differ, the ratio V/K_m is the same for all amino acid substrates. Thus an equimolar mixture of amino acids will give rise to an equimolar mixture of products. The kinetic properties of the enzyme are consistent with a partially ordered mechanism with arginine (NADPH, then arginine or pyruvate). Octopine synthase is a monomeric enzyme with a molecular weight of 39,000 by gel filtration and 38,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Synthesis of Amino Acids from Hydrocarbons

Isolation of microorganisms capable of synthesising amino acids, utilizing hydrocarbons, has been reported. These microorganisms were isolated from soil samples by selective culture techniques. 91 strains were found capable of producing amino acids in the broth. Different amino acids and their maximum yield obtained were glutamic acid 160 mg/1; leucine 90.0 mg/1; isoleucine 40.0 mg/1; valine 105.0 mg/1; methionine 25.0 mg/1; tryptophan 2.5 mg/1; arginine 70.0 mg/1; and histidine 10.0 mg/1.     

肝硬化疾病与支链氨基酸应用研究进展

蛋白质-营养不良是肝硬化病人最常见的并发症之一。肝脏作为蛋白质、脂类和糖代谢的主要器官,病变后的代谢紊乱随之而来。不适宜的蛋白质-能量摄入只会加重病情最后发生肝性脑病等危及生命的严重后果。因此,肝硬化病人的营养管理显得尤为重要,氨基酸的适宜供给无疑是营养治疗的重中之重。已知支链氨基酸能通过刺激肝细胞合成、减少肝损伤后的分解代谢等诸多方式改善营养状况,但是各种试验结果仍存在争议。最佳适宜量究竟多大,安全性范围的设定以及确切的保护机理等问题仍待进一步深入研究。     

Proton NMR Spectra of 2,4-Dinitrophenyl Derivatives of Amino Acids

During the decay of wood by the typical white rot fungus Coriolus versicolor, Laccase III was the most abundantly secreted phenol oxidase. In this study, we proposed a possibility of the intermediate degradation steps of polymeric lignin by a purified Laccase III using synthetic [β-¹³C] and [β-¹⁴C]lignin (DHP). When the [β-¹⁴C]DHP was incubated with Laccase III, the water-soluble degradation product formed was about 8% of the applied [β-¹⁴C]DHP. The enzymic attack of Laccase III catalyzed the cleavage of the intermonomer linkages in the side chain structure of the polymeric lignin. In polymeric lignin metabolism by this fungus, laccase activity was closely related to the accumulation of water-soluble degradation products.     

Extracellular Overflow of Neuroactive Amino Acids During Severe Insulin-Induced Hypoglycemia: In Vivo Dialysis of the Rat Hippocampus

Hypoglycemia-evoked changes in levels of extracellular excitatory and inhibitory amino acids were studied using the microdialysis technique. A newly designed dialysis probe was inserted stereotaxically into the rat hippocampus. Animals were then subjected to insulin-induced hypoglycemia; then blood glucose levels were restored by glucose injections after a 30-min period of isoelectric electroencephalography. Dialysates were collected before, during, and after the isoelectric period. Amino acids in the dialysates were analyzed by liquid chromatography and fluorescence detection following automatic precolumn derivatization with o-phthaldialdehyde. During the isoelectric phase, the concentration of aspartate increased 15-fold, whereas glutamate, gamma-amino-butyric acid, taurine, and phosphoethanolamine levels were elevated three- to sixfold. Smaller increases were observed for nonneuroactive amino acids such as asparagine, alanine, and phenylalanine. In contrast to all other amino acids, the glutamine content was reduced to less than 30% of preisoelectric values. The concentrations of the neuroactive amino acids were restored to normal in the post-isoelectric phase. These data demonstrate that there is an extracellular overflow of neuroactive amino acids, especially aspartate, during severe hypoglycemia.     

Synthesis of Photolabile Caged Amino Acids for Measuring Amino Acid Transporters

Photolabile precursors (caged compounds) of amino acids such as Ala, Leu, Lys, and Ser were prepared by some simple reactions. These compounds were designed for the rapid, photochemically initiated release of amino acids. These amino acid transporters were expressed in Xenopus oocyte by injecting mRNA prepared from rat kidney. The electrical response of each transporter was examined by applying the amino acids and caged compounds before and after photolysis. Photolysis of the caged amino acids increased the electrical response of the facilitated amino acid transporters expressed in the oocyte. Consequently, these synthesized caged amino acids would be applicable to kinetic investigations on the transporters when combined with a pulsed laser or xenon arc flash lamp.     

Caffeic Acid Derivatives: In Vitro and In Vivo Anti-inflammatory Properties

Objective: This study examined whether, daily fruit (blueberries) consumption (250 g) for three weeks or acute fruit ingestion (250 g) would attenuate angiotensin converting enzyme (ACE) activity and reduce oxidative stress in chronic cigarette smokers.

Methods: Twenty subjects were recruited and randomized into fruit or control groups. Blood samples and blood pressure were obtained at baseline and then pre and one hour post when subjects returned to the lab three weeks later. To examine acute effects, the fruit group immediately ingested 250 g of blueberries after returning and at least one hour prior to the post blood draw. Plasma samples were analyzed for ACE activity, F₂- isoprostanes and lipid hydroperoxides (LH) as measures of oxidative stress, and ferric reducing ability of plasma (FRAP) as a measure of antioxidant potential. A 2 (treatment) × 3 (time) repeated measures ANOVA was used for statistical analysis. If interaction was significant, then Student's t-tests were used to further examine this relationship. For these comparisons, a Bonferroni adjustment was made with statistical significance set at P < 0.025.

Results: The pattern of change between treatments was not significant for any variable except LH (P < 0.001).

Conclusion: This study indicates that LH are significantly reduced by daily fruit consumption, but not affected by acute ingestion. This finding could be one way in which fruit consumption contributes to prevention of cardiovascular disease.     

Agrobacterium tumefaciens Ribonucleic Acid Synthesis in Tomato Cells and Crown Gall Induction

Purified Agrobacterium tumefaciens deoxyribonucleic acid (DNA) does not produce crown gall tumors in growing plants, conditioned by wounding, as the living bacteria do. Purified bacterial DNA migrates in the plant and replicates, but it is not transcribed in our experimental conditions. On the contrary, when DNA is released naturally from bacteria into plant cells, a bacterial ribonucleic acid (RNA) can be found in these cells. There seems to be a direct relation between the appearance of A. tumefaciens RNA in the plant cells and the induction of the tumor.     

Effect of Amygdaloid Kindling on the Content and Release of Amino Acids from the Amygdaloid Complex: In Vivo and In Vitro Studies

Abstract: The tissue content and the interstitial fluid levels of glutamate, aspartate, GABA, glutamine, glycine, and serine were studied in amygdaloid-kindled rat brain. Interstitial levels were studied in vivo before and during stage 5 full limbic seizures using microdialysis. Slices of amygdala from kindled and sham-operated animals were used to study baseline and KCl-evoked release in vitro. The contents of these amino acids were measured in slices of amygdala, hippocampus, and cerebral cortex from kindled and sham-operated animals. Kindled brains showed two- to threefold higher levels of glutamate, aspartate, and GABA and 12-fold higher levels of glutamine than sham-operated controls. Correlating with this, interstitial fluid levels of glutamate were two- to threefold higher from kindled amygdala than from control both in vivo (microdialysis) and in vitro (superfusion). GABA levels in interstitial fluid from kindled amygdala were reduced by 67% compared with control amygdala.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

Saturable Retention of Vasopressin by Hippocampus Vessels In Vivo, Associated with Inhibition of Blood-Brain Transfer of Large Neutral Amino Acids

Vasopressin receptors have been reported in the endothelium of brain capillaries. The function of these receptors is not known. To test the prediction that vasopressin receptors in brain capillary endothelium affect amino acid transport across the blood-brain barrier and to assess the role of vasopressin transport across the cerebral vascular endothelium, we measured (a) the endothelial permeability to the large neutral amino acid leucine in the absence and presence of arginine vasopressin (AVP) and (b) the permeability of the blood-brain barrier to AVP relative to manitol. In brain regions protected by the blood-brain barrier, after circulation for 20 s, coinjection of leucine and AVP intravenously led to a decrease of leucine transport unrelated to changes of blood flow. The decrease was most pronounced in hippocampus (42%) and least pronounced in olfactory bulb and colliculi (17 and 19%, respectively). In the latter regions, the endothelial permeability to AVP did not significantly exceed that of mannitol. In hippocampus and in regions with no blood-brain barrier (pituitary and pineal glands), AVP retention in excess of mannitol retention was blocked by unlabeled AVP. The findings do not contradict the hypothesis of a role for AVP in the regulation of large neutral amino acid transfer into brain tissue.