Characteristics of the Large Neutral Amino Acid Transport System of Bovine Brain Microvessel Endothelial Cell Monolayers

The large neutral amino acid (LNAA) transport system, or L (leucine) system, in primary cultures of bovine brain microvessel endothelial cell monolayers has been characterized. The transendothelial transport of leucine in this in vitro blood-brain barrier (BBB) model was determined to be bidirectional and time, temperature, and concentration dependent. Leucine transport was saturable, and the apparent Km and Vmax were determined to be 0.18 mM and 6.3 nmol/mg/min, respectively. Transendothelial transport of leucine was resistant to inhibition by ouabain and sodium azide. Other LNAAs, including the centrally acting drugs alpha-methyl-3,4-dihydroxyphenylalanine (alpha-methyldopa), L-3,4-dihydroxyphenylalanine (L-DOPA), alpha-methyltyrosine, and baclofen, inhibited leucine transport. The leucine carrier system was also found to be stereospecific. Sucrose, used as a diffusion marker, moved across the monolayers slowly, and its concentration was not significant for at least 30 min.     

Inhibition of Large Neutral Amino Acid Transporters Suppresses Kynurenic Acid Production Via Inhibition of Kynurenine Uptake in Rodent Brain

The tryptophan metabolite, kynurenic acid (KYNA), is a preferential antagonist of the α7 nicotinic acetylcholine receptor and N-methyl-d-aspartic acid receptor at endogenous brain concentrations. Recent studies have suggested that increases of brain KYNA levels are involved in psychiatric disorders such as schizophrenia and depression, and regulation of KYNA production has become a new target for treatment of these diseases. Kynurenine (KYN), the immediate precursor of KYNA, is transported into astrocytes via large neutral amino acid transporters (LATs). In the present study, the effect of LATs regulation on KYN uptake and KYNA production was investigated in vitro and in vivo using an LATs inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). In the in vitro study, cortical slices of rat brain were incubated with a physiological concentration of KYN and 3 µmol/L–3 mmol/L BCH. BCH inhibited KYNA production and KYN uptake in a dose-dependent manner, and their IC₅₀ values were 90.7 and 97.4 µmol/L, respectively. In the in vivo study, mice were administered KYN (50 mg/kg BW) orally and BCH (200 mg/kg BW) intravenously. Administration of KYN increased brain KYN and KYNA levels compared with the mice treated with vehicle, whereas additional administration of BCH suppressed KYN-induced elevations in KYN and KYNA levels to 50 and 70 % in the brain. These results suggest that inhibition of LATs prevented the increase of KYNA production via blockade of KYN uptake in the brain in vitro and in vivo. LATs can be a target to modulate brain function by regulation of KYNA production in the brain.     

Effects of Mitochondrial Toxins on the Brain Amino Acid Concentrations

In the pathogenesis of Parkinson’s disease and Huntington’s disease excitotoxicity may play an important role. The common toxin model for Parkinson’s disease is MPTP, while for Huntington’s disease it is 3-NP. These toxins inhibit the mitochondrial respiratory chain, resulting in an energy deficit. In the central nervous system, the amino acids act as neurotransmitters and neuromodulators. The energy deficit caused by these neurotoxins may alter the concentrations of amino acids. Thus, it can be claimed that the aminoacidergic neurotransmission can be changed by neurotoxins. To test this hypothesis we studied the amino acid concentrations in different brain regions following MPTP or 3-NP administration. The two toxins were found to produce similar changes. We detected marked decreases in most of the amino acid concentrations in the striatum and in the cortex, while the levels in the cerebellum increased significantly. The decreased amino acid levels can be explained by the reduced levels of ATP produced by these neurotoxins. In the cerebellum, where there is no detectable ATP loss, the elevated amino acid levels may reflect a compensation of the altered neurotransmission.     

氨基酸分析法快速测定梭曼中毒后大鼠脑组织中兴奋性氨基酸的含量

建立一种快速、准确、可靠的脑内兴奋性氨基酸定量检测方法 ,并观察梭曼惊厥后大鼠脑组织中兴奋性氨基酸(EAAs)含量变化。采用 6 30 0黄金系统氨基酸分析仪 ,在锂柱 130min程序生理体液分析方法基础上 ,根据兴奋性氨基酸(EAAs)的特性 ,建立了EAAs的快速测定方法 ,并用此方法对梭曼惊厥后不同时相大鼠的新鲜脑组织进行定位检测。梭曼诱发惊厥后大脑皮质和海马内谷氨酸和天冬门氨酸水平显著下降。惊厥 30min时谷氨酸下降最明显 ,分别是正常组的 5 3.2 %和 5 2 .8%。天门冬氨酸更易受梭曼中毒的影响 ,惊厥后 5、30、90min 3个时相点测定值均显著下降。此方法完成谷氨酸和天门冬氨酸分析的时间是 2 0min ,比原方法缩短了 110min ;且有较好的重现性 (GluCV :日内 1.86 % ,日间 2 .32 % ;AspCV :日内 1.42 ,日间 2 .48% )和回收率 (Glu 97.7% ;Asp97.3% )。兴奋性氨基酸参与了梭曼中毒性惊厥的病理生理过程。本方法定量检测兴奋性氨基酸快速、准确 ,并利于大批量样品的快速测定     

慢性肾炎体液氨基酸与正常比较

慢性肾炎体液氨基酸与正常比较蒋滢,黄美英,何达纯ＣｈａｎｇｅｏｆＦｌｕｉｄＦｒｅｅＡｍｉｎｏＡｃｉｄｉｎＰａｔｉｅｎｔｓＷｉｔｈＣｈｒｏｎｉｃＮｅｐｈｒｉｔｉｓ￥ＪｉａｎｇＹｉｎｇ;ＨｅＤａｃｈｕｎａｎｄＨｕａｎｇＭｅｉｙｉｎｇ（Ｄｅｐａｒｔｍｅｎｔ...     

Studies on Amino Acid Metabolism in the Brain Using 15N-Labeled Precursors

The transfer of label from ¹⁵N-alanine and ¹⁵N-glutamate into amino acids in incubated brain slices has been followed using gas chromatography/mass spectrometry (GC/MS). ¹⁵N from alanine appeared in both amino and amide groups of glutamine more rapidly than into aspartate, glutamate and GABA, which were all labeled at similar rates. Maximum labelling of approx. 50% enrichment of these three metabolites was achieved in 3 hr. The ¹⁵N present in doubly-labeled glutamine exceeded that in the singly-labelled after 30 min. ¹⁵N from glutamate was rapidly transferred to aspartate and to alanine, with slower incorporation into glutamine and GABA. As was seen with labeling from alanine, doubly-labeled glutamine was higher than the singly-labeled species, also reaching some 50% enrichment in 3 hr. Depolarisation with 40 mM extracellular K⁺ caused a considerable reversal of the ratio of doubly- to singly-labeled glutamine species from both alanine and glutamate. The results are discussed in terms of the effects of depolarization on the glutamate/glutamine cycle.     

Physicochemical Properties and Amino Acid Sequence of Sheep Brain Galectin-1

A beta-galactoside-specific soluble 14-kD lectin from sheep brain was isolated, sequenced, and compared with similar galectins from other species. Percent identities of amino acid sequence and the carbohydrate recognition domain (CRD) revealed that the isolated galectin shares all the absolutely preserved and critical residues of the mammalian galectin-1 subfamily. The isolated sheep brain galectin (SBG) showed more than 90% amino acid sequence (92%) and carbohydrate recognition domain identity (96%) with human brain galectin-1. Conformational changes were found induced by interaction of the protein with its specific disaccharide and oxidizing agent (hydrogen peroxide). Upon oxidation a drastic change in the environment of aromatic residues and conformation of the galectin was observed with the loss of hemagglutination activity, while no significant change was observed upon addition of D-lactose (Gal(beta1-4)Glc) in the far-UV and near-UV spectra, suggesting no significant modification in the secondary as well as tertiary structures of sheep brain galectin. But the functional integrity of the CRD is found to be affected in the presence of oxidizing agent, indicating intramolecular disulfide bonds and requirement of complete polypeptide chain for functional integrity of the carbohydrate recognition domain.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

脑脊液分流术治疗64例脑肿瘤临床效果分析

目的:通过观察脑脊液分流术在64例脑肿瘤患者中的临床应用效果,探讨其临床应用的价值。方法:收集2009年1月-2010年3月我院64例脑肿瘤患者病例资料,其临床诊断均有颅内高压,对照组32例患者给予常规手术进行肿瘤切除;观察组在对照组的基础上给予脑脊液分流术,比较两组治疗效果。结果:治疗后两组颅内压均降低,观察组降低更为明显(P0.05)。对照组完全缓解率为15.6%,部分缓解率为21.8%,病程稳定率为31.3%,病程进展率为31.3%。观察组分别为28.1%、31.3%、25%和15.6%。观察组完全缓解率和部分缓解率明显高于对照组(P0.05.),病程进展率对照组明显高于观察组(P0.05)。对照组术后两年内复发5例,生存超过三年的17例,生存超过五年的12例。观察组术后两年内复发的2例,生存超过三年的20例,生存超过5年的15例。和对照组比较,观察组术后病情复发率更低,生存指数更高,比较有明显差异(P0.05)。结论:脑脊液分流术在伴颅内高压或脑积水的脑肿瘤手术中的使用效果显著,后期的手术成功率和患者生存率提高,临床上可予以更为深入的探索。     

Identification of Multiple Novel Protein Biomarkers Shed by Human Serous Ovarian Tumors into the Blood of Immunocompromised Mice and Verified in Patient Sera

The most cancer-specific biomarkers in blood are likely to be proteins shed directly by the tumor rather than less specific inflammatory or other host responses. The use of xenograft mouse models together with in-depth proteome analysis for identification of human proteins in the mouse blood is an under-utilized strategy that can clearly identify proteins shed by the tumor. In the current study, 268 human proteins shed into mouse blood from human OVCAR-3 serous tumors were identified based upon human vs. mouse species differences using a four-dimensional plasma proteome fractionation strategy. A multi-step prioritization and verification strategy was subsequently developed to efficiently select some of the most promising biomarkers from this large number of candidates. A key step was parallel analysis of human proteins detected in the tumor supernatant, because substantially greater sequence coverage for many of the human proteins initially detected in the xenograft mouse plasma confirmed assignments as tumor-derived human proteins. Verification of candidate biomarkers in patient sera was facilitated by in-depth, label-free quantitative comparisons of serum pools from patients with ovarian cancer and benign ovarian tumors. The only proteins that advanced to multiple reaction monitoring (MRM) assay development were those that exhibited increases in ovarian cancer patients compared with benign tumor controls. MRM assays were facilely developed for all 11 novel biomarker candidates selected by this process and analysis of larger pools of patient sera suggested that all 11 proteins are promising candidate biomarkers that should be further evaluated on individual patient blood samples.     

Methionine Sulfoximine Prevents the Accumulation of Large Neutral Amino Acids in Brain of Portacaval-Shunted Rats

Portal-systemic shunting and hyperammonemia lead to an accumulation of the large neutral amino acids in brain and apparently alter transport of neutral amino acids across the blood-brain barrier. It has been proposed that portal-systemic shunting leads to a high brain concentration of glutamine, a product of cerebral ammonia detoxification, and thereby affects the transport of other neutral amino acids across the blood-brain barrier. To test this hypothesis, rats with a portacaval shunt were treated with L-methionine-dl-sulfoximine (MSO), an inhibitor of glutamine synthesis. Treatment with MSO resulted in lower concentrations of the neutral amino acids in brain of portacaval-shunted rats and a higher brain ammonia concentration, compared with untreated shunted rats. These results suggest that the accumulation of neutral amino acids in brain after portacaval shunt depends on the increased synthesis of glutamine in brain.     

The Deletion of Several Amino Acid Stretches of Escherichia coli Alpha-Hemolysin (HlyA) Suggests That the Channel-Forming Domain Contains Beta-Strands

Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyA_Δ71–110 and HlyA_Δ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyA_Δ158–167 and HlyA_Δ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyA_Δ71–110 and HlyA_Δ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyA_Δ71–110, and HlyA_Δ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.     

衰老大鼠的某些脑区组织中游离氨基酸水平的改变

使用D 半乳糖建立衰老大鼠模型组与同龄、同饲的正常对照组大鼠的某些脑区游离氨基酸 (FAA)水平的比较发现 :( 1 )衰老模型组的海马、纹状体以及皮层等脑区中谷氨酸 (Glu)、天门冬氨酸 (Asp)水平明显降低 ;( 2 )γ 氨基丁酸 (GABA)水平在衰老模型组大鼠的海马 ,纹状体以及小脑等脑区中明显升高 ;( 3)衰老模型组的皮层、小脑、海马、纹状体等脑区的牛磺酸 (Tau)水平明显下降。以此探讨动物衰老与脑区游离氨基酸水平的关系     

Abnormalities in Amino Acid Neurotransmitters in Discrete Brain Regions of Genetically Dystonic Hamsters

The concentrations of 11 amino acids, including the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, glycine, and taurine, were determined by HPLC in 12 brain regions of genetically dystonic (dtSZ) hamsters and age-matched nondystonic controls. Since dystonia in mutant dtSZ hamsters is transient and disappears after about 70 days of age, amino acids were determined at the age of maximum severity of dystonia (30-40 days) and after disappearance of the disease, to examine which neurochemical changes were related to dystonia. In dtSZ hamsters with the maximum severity of dystonia, significant changes in concentrations of the neurotransmitters gamma-aminobutyric acid, glutamate, aspartate, and taurine were found in several regions involved in motor functions, e.g., cerebellum, thalamus, and corpus striatum. Most of these changes were not permanent but disappeared in parallel with dystonia, implicating a causal relationship between altered aminoacidergic neurotransmission and dystonia in mutant dtSZ hamsters.     

Mechanisms of Inhibitory Amino Acid Release in the Brain Stem Under Normal and Ischemic Conditions

This article deals with the release of GABA, glycine and taurine from the brain stem under normal conditions and in ischemia. The release mechanisms, the effects of glutamate and adenosine receptors, and the roles of nitric oxide and second messengers are reviewed.     

615小鼠血红蛋白α链的氨基酸组成及个别肽段的氨基酸序列

用CM-Cellulose-23柱层析分离纯化了615小鼠珠蛋白α链,测定其N端氨基酸残基为缬氨酸.615小鼠珠蛋白α链含有141个氨基酸残基,其中19个亮氨酸残基,10个组氨酸残基,9个缬氨酸残基,上述氨基酸残基的数目与文献中其亲本C₅₇BL不同.用胰蛋白酶水解615小鼠珠蛋白α链,发现有不溶性的‘核心’和可溶性的酶解片段.其中一个酶解肽段从N端数第8位氨基酸残基发生了突变,由亲本的缬氨酸变为亮氨酸.     

Multiple Subtypes of Excitatory Amino Acid Receptors Coupled to the Hydrolysis of Phosphoinositides in Rat Brain

Abstract: The excitatory amino acid (EAA) analogues quisqualate, ibotenate, and trans-(±)-1-amino-1, 3-cyclopentanedicarboxylate (trans-ACPD) activate the metabotropic EAA receptors that are coupled to the hydrolysis of Phosphoinositides (PI). Previous studies of hippocampal cross sections demonstrated that PI hydrolysis stimulated by these agonists can be inhibited by either L-aspartate-β- hydroxamate (L-AβHA) or DL-2-amino-3-phosphonopropionate (DL-AP3). The goal of the present studies was to determine if all metabotropic EAA receptors are sensitive to L-AβHA and DL-AP3. Two approaches were used. In the first, using cerebellar cross sections, the effects of these agonists and inhibitors were examined. The EC₅₀ values (the concentrations required to evoke half-maximal stimulation) of quisqualate, ibotenate, and trans-ACPD in cerebellum were similar to the EC₅₀ values that we observed previously in hippocampus, but neither L-AβHA nor DL- AP3 blocked PI hydrolysis. The EC₅₀ values were 0.65 ± 0.17 μM for quisqualate, 12.8 ± 2.5 μM for ibotenate, and 18.1 ± 3.1 μM for trans-ACPD. All data were best fit to theoretical curves that had Hill slopes of 1. In the second approach, another EAA analogue, D-aspartate, was identified as an agonist that stimulates PI hydrolysis. The EC₅₀for PI hydrolysis stimulated by D-aspartate was 470 ± 90 μM in hippocampus. Neither L-AβSHA nor DL-AP3 blocked PI hydrolysis stimulated by D-aspartate in hippocampus. Furthermore, antagonists of ionotropic EAA receptors, antagonists of other receptor systems coupled to PI hydrolysis, and inhibitors of the Na⁺-dependent L-glutamate transport process also did not block PI hydrolysis stimulated by D-aspartate. These data support the presence of three pharmacologically distinct metabotropic EAA receptor subtypes.