Free Radicals and the Ischemia-Evoked Extracellular Accumulation of Amino Acids in Rat Cerebral Cortex

The effects of free radical generating systems on basal and ischemia/reperfusion-evoked release of amino acids into cortical superfusates was examined in the rat using the cortical cup technique. Xanthine oxidase plus xanthine significantly enhanced GABA levels 358 fold over controls during 20 min of four vessel occlusion. Glutamate and phosphoethanolamine release following reperfusion were also elevated. Prostaglandin synthase plus arachidonic acid significantly enhanced the ischemia-evoked release of all amino acids (aspartate 360 fold; glutamate 433 fold; glycine 6 fold; GABA 689 fold; phosphoethanolamine 69 fold) and increased the pre-ischemic levels of glutamate, glycine and phosphoethanolamine. Administration of H₂O₂ plus ferrous sulfate significantly elevated both pre-ischemic amino acid release and ischemia-evoked release. A role for free radical generating systems in the development of ischemic injury is supported by the ability of superoxide dismutase plus catalase to reduce ischemia-evoked amino acid efflux into cortical superfusates. Thus, the species of free radical produced, as well as the amount generated, may alter the pattern of amino acid release under both ischemic and non-ischemic conditions.     

不同品种药用菊花中游离糖类及游离氨基酸含量的HPLC分析

为比较不同品种菊花的品质特征,本文采用HPLC法测定其游离糖类及游离氨基酸的含量.结果表明,不同品种菊花中游离糖类主要成分是果糖和葡萄糖,蔗糖和麦芽糖未检出,总游离糖类含量约为9.07～18.76g/1O0g,其中黄山贡菊中含量最高,为18.76g/100g,黄菊中最低,为9.07g/100g;其总游离氨基酸含量约为0.563～2.806g/100g,七月菊中含量最高,为2.806g/100g,杭白菊中最低,为0.563g/100g.     

外源氨基酸胁迫下植物体内游离氨基酸的测定

目的：通过检测外源氨基酸胁迫下植物体内的游离氨基酸含量,探讨氨基酸胁迫下植物生长受抑制的机制。方法：小麦胚接种在加入D-丙氨酸、D-丝氨酸和L-缬氨酸等3种外源氨基酸的培养基中,其发芽和生长会受到强烈抑制。取材胁迫植株和对照植株,提取并用HPLC方法检测游离氨基酸含量,分析主要氨基酸特征的改变。结果：作为处理的胁迫氨基酸在体内有数倍到数十倍的增高,其他氨基酸,尤其是同族及相近族氨基酸的量也出现较大改变,有增加也有减少,有些氨基酸甚至检测不出。结论：在外源氨基酸胁迫下,植物可直接吸收这些氨基酸,胁迫氨基酸在体内的积累。至少影响到了部分氨基酸的合成,使细胞内正常游离态氨基酸的数量增加或减少,这些变化引起代谢失调,进而引起生长抑制。     

Formation of a Precursor of the Free Radical Species in the Reaction of Dehydroascorbic Acid with Amino Acids

A precursor of a known long life free radical species which gives a triplet ESR signal was detected among the reaction products of dehydroascorbic acid and amino acids. Experimentally, this compound was transformed to the radical species by dilution with 1: 1 pyridinewater or some buffer solutions of pH 5~7. The yield of the precursor was significantly improved by the addition of ascorbic acid to the reaction system.     

Variability for Free Sugars and Organic Acids in Capsicum chinense

Variability in the concentrations of the chemical constituents that contribute to fruit flavor in Capsicum chinense is poorly documented in the scientific literature. We surveyed fruit of 216 landraces and cultivated varieties of Capsicum chinense acquired from North, Central, and South America, and analyzed these for concentrations of the simple sugars sucrose, glucose, and fructose, and citric, malic, succinic, fumaric, and ascorbic acids. Concentrations (mg/100 g Fresh Weight (FW) of whole fruit) of sucrose, glucose, and fructose in fruit of this species ranged from 0.0 to 150, 68 to 701, and 101 to 823, respectively. The total of these sugars (sucrose+glucose+fructose) ranged from 198 to 1543 mg/100 g FW. Concentrations of organic acids ranged from 0.0 (not detected) to 818, 430, 340, and 232 mg/100 g FW for citric, malic, fumaric, and succinic acids, respectively. However, the relative ranking in the concentrations of the individual acids was genotype‐dependent. Total ascorbic‐acid values ranged from 30 to 1466 mg/100 g FW. These data serve to document the range in the concentrations of individual sugars and acids present in mature C. chinense fruit and suggest that this variability may lend itself to studies involving the synthesis and/or metabolism of compounds associated with fruit flavor.     

A New Enzymatic Microdetermination Procedure for Ethanol with Particulate Alcohol Dehydrogenase from Acetic Acid Bacteria

A new enzymatic method for microdetermination of ethanol has been established with particulate alcohol dehydrogenase from acetic acid bacteria and applied to the practical purposes. The enzyme had an optimum pH for ethanol oxidation at a fairly acidic region. Trace amounts of ethanol could be assayed by measuring the initial reaction rate as successful as by reading the end point of the reaction. Some advantages in using this enzyme for ethanol determination were pointed out comparing with NAD-linked alcohol dehydrogenase from yeast or horse liver. Impurity in the enzyme preparations, stability of reagents and coexistence of other substances in the assay mixture were not as critical as in NAD-linked enzyme. Acidic samples could also be directly determined for ethanol without preadjustment of sample pH.     

测定荔枝核中的游离氨基酸

采用氨基酸自动分析仪测定荔枝核水提取液、醇提取液样品。在醇提取液中游离氨基酸的含量高于水提取液,在两种提取液中共检出了21种游离氨基酸(其中4种为未知氨基酸,6种必需氨基酸);已知游离氨基酸的质量分数为307μg.kg-1(醇提取液)和269μg.kg-1(水提取液),总游离氨基酸的质量分数为500μg.kg-1,必需氨基酸的量占总游离氨基酸量的44%。结论:荔枝核中的游离氨基酸具有利用价值。     

Effect of Sugars and Amino Acids on Androgenesis of Cucumis sativus

The effects of sugars (sucrose, maltose, glucose and fructose) and amino acids (glutamine, glycine, arginine, asparagine and cysteine) on embryogenesis and plantlet regeneration from cultured anthers of Cucumis sativus L. cv. Calypso and Green Long were studied. Type and concentration of sugar and amino acid influenced embryogenesis. Among the different sugars tested, sucrose was the best for embryo induction with an optimal concentration of 0.25 M. Maximum of 72 and 80 embryos per 60 anthers of Calypso and Green Long, respectively, were induced on embryo induction medium [B5 (Gamborg, Miller and Ojima (1968) Exp. Cell Res. 50: 151–158) supplemented with 2.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 μM 6-benzyladenine (BA)] containing 0.25 M sucrose. The addition of amino acids to the embryo induction medium improved embryo yield with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) giving the best response. Embryo differentiation was achieved on B5 medium supplemented with 0.25 μM of α-naphthaleneacetic acid (NAA), 0.25 μM kinetin (KN) and 0.09 M sucrose. Embryos were converted on B5 medium supplemented with abscisic acid (ABA) (10 μM) and 0.09 M sucrose. Embryos that developed on B5 medium supplemented with a combination of amino acids (glutamine, glycine, arginine, asparagine and cysteine of 1.0 mM each) exhibited the highest plantlet regeneration frequency.     

苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

苏云金芽胞杆菌转座因子的转座机理及其与杀虫晶体蛋白基因的关系

各种苏云金芽胞杆菌在杀虫毒力和杀虫谱上有很大差异。研究表明,这种特异性的杀虫毒力与存在于苏云金芽胞杆菌内的转座因子有密切关系,不同类型的转座因子其转座方式各异,总的来说可分为３种,即同源重组、转座重组和特异位点重组。这种转座过程的发生往往伴随着苏云金芽胞杆菌杀虫晶体蛋白的变异,这在基因工程菌的构建和杀虫多样性的研究上有着重要意义。     

Electrogenic Absorption of Sugars and Amino Acids in the Small Intestine of the Human Fetus

Some characteristics and the degree of intestinal absorption in the developing human fetus were examined by measuring solute evoked potentials and ¹⁴C-D-glucose uptake into the everted jejunal segments.

In all segments, the Michaelis-Menten relationship was observed between D-glucose concentrations and the evoked potentials or D-glucose uptake. Increase of Na-ion concentrations enhanced both D-glucose evoked potentials and uptake.

Both D-glucose and L-α-alanine evoked potentials increased in a significant correlation to the fetal age; however, the apparent Michaelis constants did not show any significant change. The structural specificity of sugar for generating evoked potentials was the same as that reported for adult mammals. Among amino acids, only the L-form of neutral and acidic amino acids generated markedly high evoked potentials, but the basic ones hardly at all. Oligopeptides such as glycyl-glycine and glycyl-glycyl-glycine also generated high evoked potentials.     

光质对水稻幼苗蛋白质、氨基酸含量的影响

蓝光对水稻幼苗可溶性蛋白积累的促进效应,在幼苗生长的初期比较明显;第５天,幼苗的可溶性蛋白质、蛋白氮、非蛋白氮以及Ａｓｐ、Ａｓｎ、Ｇｌｕ、Ｇｌｎ等游离氨基酸含量都远远高于白光或红光的处理。随着苗龄的增加,蓝光的促进作用减弱,到第１７天,各项指标都低于白光处理的幼苗。红光处理的幼苗可溶性蛋白始终低于白光或蓝光的处理,其Ａｓｎ、Ｇｌｎ两种酰胺含量在第１０天以后明显高于同期的白光或蓝光的处理。     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N 乙酰高丝氨酸内酯 (N acyl homoserinelactones,AHLs) ,是一类数量感知 (Quorum sensing)系统中的信号分子 ,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子 ,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子 ,它相对于其它cry类基因的启动子有启动基因转录时间早 ,转录时间长的优点。通过重叠延伸PCR ,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子 ,构建了融合基因pro3A aiiA。将融合基因装入穿梭载体pHT3 0 4的BamHI SphI位点 ,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株 ,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株     

Subcellular Distribution of Free Amino Acids in Relation to Protein Synthesis in Cells of Chara corallina

The influence of protein-synthesis inhibitors on the subcellular distribution of free amino acids was studied in internodal cells of Chara corallina. Use of an intracellular perfusion technique allowed separate measurements of amino acids in the vacuole, in the flowing sol endoplasm and in the gel layer. The sol endoplasm predominantly represents the cytosol, while the gel layer is occupied, for the most part, by chloroplasts. When cells were treated with 0.5 mM chloramphenicol (CRP) in the dark, both the total concentration of amino acids and the subcellular distribution were almost the same as in cells without treatment. In the light, however, the subcellular distribution changed dramatically, although the total concentration of amino acids was unchanged. The vacuolar concentration of amino acids was 3 times greater in CRP-treated cells than in the control. The concentrations of amino acids in the sol endoplasm and in the gel layer were only half of those in the control. Amino acid permeability of the chloroplast envelope, measured using the perfused internodal cells, slightly increased after the CRP treatment in the light. Time-dependent changes in concentrations of amino acids in the CRP-treated cells were also measured in the light. The total concentration of amino acids in the cytoplasm gradually decreased, while that in the vacuole increased commensurately. The concentration and/or subcellular distribution of alanine, glutamine, glutamate and glycine changed dramatically. The concentration of alanine increased considerably both in the vacuole and in the cytoplasm. The cytoplasmic concentration of glutamine increased transiently within 1 ?2 h after treatment with CRP. The cytoplasmic concentrations of glutamate and glycine decreased. Although the concentrations of some amino acids changed so markedly both in the vacuole and cytoplasm, only small differences in the activities of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and glutamine synthetase were detected between the control and the CRP-treated cells.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

The use of N-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite the tert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of the N-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Side Reaction with N-Carboxymethyl Amino Acids in the Synthesis of Backbone Cyclized Peptides

Summary The use ofN-carboxymethyl amino acids in the assembly of peptides with backbone cyclization can lead to diketopiperazine formation by intramolecular aminolysis which occurs despite thetert-butyl protection of the carboxy group. This undesired side reaction can be prevented by a very short deprotection time for the Fmoc group, by elongation of theN-carboxyalkyl chain or by forming the backbone (lactam) bridge before Fmoc removal, but not by the use of DBU or additives.     

Cloning and expression of the cry1Ea4 gene of Bacillus thuringiensis and the comparative toxicity of its gene product

A new cry gene (cry1Ea4) was cloned and sequenced from a Bacillus thuringiensis isolate native to Mexico (LBIT-147). The gene coded for a 133kDa protoxin which had greater than 99% homology with the holotype Cry1Ea1, as only four mismatches were found between the two amino acid sequences. When the Cry1Ea4 toxin was expressed in a crystal-negative strain of B. thuringiensis, bipyramidal crystals were produced. Purified crystals from this recombinant strain and from the holotype (Cry1Ea1) were bioassayed against first instar larvae of the tobacco hornworm. Statistically different mean LC₅₀ values indicated that Cry1Ea4 was more toxic than its holotype. This increase in toxicity may be attributed to the three amino acids which differ from the holotype sequence in the toxic fragment.     

Changes in Volatile Fatty Acids and Free Amino Acids during Air-Tobacco Stalks

In our searching program for novel sorbicillin related compounds, three novel compounds, spirosorbicillinols A (1), B (2), and C (3), were isolated from the fermentation broth of the USF-4860 strain isolated from a soil sample. The planar structures of compounds 1–3 were determined from spectroscopic evidence and degradation reaction, and that of 1 was the same as that of 2. The relative stereochemistries of compounds 1–3 were determined by ¹H-¹H coupling constants, the elucidation of HMBC and NOESY spectra in detail. 1 and 2 were stereoisomers at C8 position, each other. We propose that compounds 1 and 2 were formed by exo and endo intermolecular Diels-Alder reaction between sorbicillinol as a diene and scytolide (proposed precursor-1) as a dienophile, respectively. Similarly, we propose that compound 3 was formed by an endo intermolecular Diels-Alder reaction between sorbicillinol and proposed precursor-2.     

真菌新型激活蛋白对Bt制剂的增效作用

激活蛋白是从交链孢属 (Alternaria)真菌分离的新型多功能蛋白 ,以棉铃虫和小菜蛾幼虫为供试昆虫 ,用饲料染毒法、浸叶法和浸虫法测定了激活蛋白对Bt的增效作用。结果表明 ,用Bt∶激活蛋白为 1∶0 0 3的含毒饲料饲喂 2龄棉铃虫幼虫 ,其增效指数为 2 2倍。Bt∶激活蛋白以 1∶0 0 95和 1∶0 6混合后分别浸叶后 ,饲喂棉铃虫和小菜蛾幼虫 ,其增效指数分别为 2 7和 5 5倍。单独用激活蛋白饲喂棉铃虫幼虫无致死活性 ,但当Bt与激活蛋白以 1∶0 75混合后 ,毒杀效果显著增强 ,对Bt的增效指数为 18 5倍。     

Distribution of Sorbitol, Neutral Sugars, Free Amino Acids, Malic Acid and Some Hydrolytic Enzymes in Vacuoles of Apple Cotyledons

Vacuoles of apple cotyledons (Malus pumila Mill. var. domesticaSchneid.) were obtained by purification with Ficoll densitygradient centrifugation after the protoplasts were lysed byboth osmotic shock and the addition of EDTA. High levels ofacid protease and carboxypeptidase activity were detected inthe vacuoles along with acid phosphatase, phosphodiesteraseand ATPase. The distribution of sorbitol and other sugars inthe vacuoles, the protoplast and extracellular free space wasdetermined. About 45, 60 and 90% of the sorbitol, glucose andsucrose, respectively, contained in whole tissue were foundin the extracellular free space, and 54% of the sorbitol inthe protoplast was detected in the vacuole. The sorbitol inthe vacuoles and the extravacuole of the protoplast was about80 and 70%, respectively, of the total sugar content of thecell. Arginine was the most abundant free amino acid in theprotoplast, and about 90% of it was contained in the vacuole.More than 80% of the total amino acids and 50% of the proteinwere also located in the vacuole, as well as most of the malate.The amounts of the total sugars, total amino acids, proteinand malate in the vacuoles were to 250, 400, 48 and 9 µg/10⁶cells, respectively. The results suggest that the vacuoles ofapple cotyledons contain a large pool of amino acids and proteinsrather than sugars, and have a close connection with proteinbody degradation. ¹ This paper is contribution A-l37 of the Fruit Tree ResearchStation. (Received January 20, 1982; Accepted May 18, 1982)