Histochemical Tests for Proteins and Amino Acids; The Characterization of Basic Proteins

For cytophysiological work it is important to have ways of demonstrating proteins and amino acids and especially of characterizing basic and non-basic proteins. The author presents a review of the more usually employed histochemical reactions for amino acids and proteic compounds in general, with several modifications which increase their sensitivity, or specificity and localization. The author describes the histochemical arginine reaction, recently introduced by him, by means of which the characterization of basic and non-basic proteins can be easily accomplished in every laboratory without costly apparatus; this reaction serves also for the demonstration of proteins in general. The application of protein histochemical tests for quantitative purposes is discussed in connection with the characterization of the basic proteins and the determination of the relative concentration and the active metabolic changes of proteic compounds.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

Stimulation of Lysine Decarboxylase Production in Escherichia coli by Amino Acids and Peptides

A commercial hydrolysate of casein stimulated production of lysine decarboxylase (EC 4.1.1.18) by Escherichia coli B. Cellulose and gel chromatography of this hydrolysate yielded peptides which were variably effective in this stimulation. Replacement of individual, stimulatory peptides by equivalent amino acids duplicated the enzyme levels attained with those peptides. There was no indication of specific stimulation by any peptide. The peptides were probably taken up by the oligopeptide transport system of E. coli and hydrolyzed intracellularly by peptidases to their constituent amino acids for use in enzyme synthesis. Single omission of amino acids from mixtures was used to screen them for their relative lysine decarboxylase stimulating abilities. Over 100 different mixtures were evaluated in establishing the total amino acid requirements for maximal synthesis of lysine decarboxylase by E. coli B. A mixture containing all of the common amino acids except glutamic acid, aspartic acid, and alanine increased lysine decarboxylase threefold over an equivalent weight of casein hydrolysate. The nine most stimulatory amino acids were methionine, arginine, cystine, leucine, isoleucine, glutamine, threonine, tyrosine, and asparagine. Methionine and arginine quantitatively were the most important. A mixture of these nine was 87% as effective as the complete mixture. Several amino acids were inhibitory at moderate concentrations, and alanine (2.53 mM) was the most effective. Added pyridoxine increased lysine decarboxylase activity 30%, whereas other B vitamins and cyclic adenosine 5′-monophosphate had no effect.     

支链氨基酸对运动大鼠氨基酸代谢和运动能力的影响

观察了支链氨基酸(BCAA)对大鼠运动能力和血清游离氨基酸代谢的影响。实验用21只雄性wistar大鼠,随机分为3组:正常组、游泳对照组和游泳补充BCAA组。2个运动组每天游泳训练1h,10天后游泳6h,观察补充BCAA对大鼠游泳运动能力和血清游离氨基酸水平的影响。实验结果表明,补充BCAA可明显提高大鼠游泳存活率,抑制血清中必需氨基酸、非必需氨基酸和总氨基酸水平升高,游泳运动后大鼠的血清中乳酸和LDH的升高幅度有所降低,抑制骨骼肌LDH活力的下降。说明补充BCAA可明显提高大鼠的运动能力,减少运动造成的蛋白质分解     

脆性组氨酸三联体与复制蛋白A相互作用关键氨基酸残基的鉴定

目的：研究脆性组氨酸三联体（Fhit）对ATR/CHK1通路的影响,在确定Fhit与复制蛋白A（RPA）存在相互作用的基础上鉴定Fhit与RPA相互作用的关键氨基酸残基,为进一步研究Fhit特异的信号通路奠定基础。方法：构建一系列Fhit缺失突变体基因Fhit1～Fhit11及6种Fhit点突变体基因,将这些基因插入含GST基因的原核表达载体中,在大肠杆菌中表达并纯化GST-Fhit1～GST-Fhit11融合蛋白、突变体GST-FhitSIYEEL、GST-FhitIY、GST-FhitEL、GST-FhitF、GST-FhitA,以及GST-FhitD融合蛋白,用GST沉降技术研究Fhit与RPA相互作用的关键氨基酸残基。结果：Fhit蛋白第112～117（SIYEEL）残基可能是Fhit与RPA相互作用的关键区域,而第114（Y）残基可能是Fhit与RPA相互作用的关键氨基酸残基。结论：确定了Fhit与RPA相互作用的关键氨基酸残基,为阐明Fhit在维持基因组完整性方面的机理提供了线索。     

Allosteric Regulation of a Protein Acetyltransferase in Micromonospora aurantiaca by the Amino Acids Cysteine and Arginine

ACT domains (amino acid-binding domains) are linked to a wide range of metabolic enzymes that are regulated by amino acid concentration. Seventy proteins with ACT-GCN5-related N-acetyltransferase (GNAT) domain organization were found in actinomycetales. In this study, we investigate the ACT-containing GNAT acetyltransferase, Micau_1670 (MaKat), from Micromonospora aurantiaca ATCC 27029. Arginine and cysteine were identified as ligands by monitoring the conformational changes that occur upon amino acids binding to the ACT domain in the MaKat protein using FRET assay. It was found that MaKat is an amino acid-regulated protein acetyltransferase, whereas arginine and cysteine stimulated the activity of MaKat with regard to acetylation of acetyl-CoA synthetase (Micau_0428). Our research reveals the biochemical characterization of a protein acetyltransferase that contains a fusion of a GNAT domain with an ACT domain and provides a novel signaling pathway for regulating cellular protein acetylation. These findings indicate that acetylation of proteins and acetyltransferase activity may be tightly linked to cellular concentrations of some amino acids in actinomycetales.     

pH Dependence of Histidine Affinity for Blood-Brain Barrier Carrier Transport Systems for Neutral and Cationic Amino Acids

The effects of pH (3.5-7.5) on the brain uptake of histidine by the blood-brain barrier (BBB) carriers for neutral and cationic amino acids were tested, in competition with unlabeled histidine, arginine, or phenylalanine, with the single-pass carotid injection technique. Cationic amino acid ( [14C]arginine) uptake was increasingly inhibited by unlabeled histidine as the pH of the injection solution decreased. In contrast, the inhibitory effect of unlabeled histidine on neutral amino acid ( [14C]phenylalanine) uptake decreased with decreasing pH. Brain uptake indices with varying histidine concentrations indicated that the neutral form of histidine inhibited phenylalanine uptake whereas the cationic form competed with arginine uptake. Since phenylalanine decreased [14C]histidine uptake at all pH values whereas arginine did not, it was concluded that the cationic form of histidine had an affinity for the cationic carrier, but was not transported by it. We propose that the saturable entry of histidine into brain is, under normal physiological circumstances, mediated solely by the carrier for neutral amino acids.     

The Effects of Copper and EDTA on the Autoxidation of Phospholipid Emulsions

Phosphatidyl ethanolamine and lecithin dispersed in water rapidly absorbed oxygen. The autoxidation was promoted with copper and inhibited with EDTA. Phosphatidyl ethanolamine was more sensitive both to copper and EDTA than lecithin. Their peroxide decomposition under the deaerated condition was promoted with copper and inhibited with EDTA. The copper-catalyzed O₂ uptake of phosphatidyl ethanolamine was lowered, when its peroxide was decreased. Thus, the mechanism of promoting the oxidation with copper may be explained as catalytic supply of initiator radicals from the peroxides by copper.     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.     

Cross-Pathway Regulation: Histidine-Mediated Control of Histidine, Tryptophan, and Arginine Biosynthetic Enzymes in Neurospora crassa

In Neurospora crassa, histidine starvation of histidine mutants resulted in derepression of histidine, tryptophan, and arginine biosynthetic enzymes. The same tripartite derepression occurred in wild-type strain 74A when it was grown in medium supplemented with 3-amino-1,2,4-triazole, an inhibitor of histidine biosynthesis. Histidine-mediated derepression of tryptophan and arginine biosynthetic enzymes was not due to a lowered intracellular concentration of tryptophan or arginine, respectively. A discussion of possible mechanisms and of similar studies in prokaryotic and eukaryotic organisms is presented.     

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839-843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (K(m)) for ATP from 76 to 1,200 muM and the K(m) for 5-formylTHF from 2.5 to 7.1 muM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.     

The Human Gene SLC25A29, of Solute Carrier Family 25, Encodes a Mitochondrial Transporter of Basic Amino Acids

The human genome encodes 53 members of the solute carrier family 25 (SLC25), also called the mitochondrial carrier family, many of which have been shown to transport carboxylates, amino acids, nucleotides, and cofactors across the inner mitochondrial membrane, thereby connecting cytosolic and matrix functions. In this work, a member of this family, SLC25A29, previously reported to be a mitochondrial carnitine/acylcarnitine- or ornithine-like carrier, has been thoroughly characterized biochemically. The SLC25A29 gene was overexpressed in Escherichia coli, and the gene product was purified and reconstituted in phospholipid vesicles. Its transport properties and kinetic parameters demonstrate that SLC25A29 transports arginine, lysine, homoarginine, methylarginine and, to a much lesser extent, ornithine and histidine. Carnitine and acylcarnitines were not transported by SLC25A29. This carrier catalyzed substantial uniport besides a counter-exchange transport, exhibited a high transport affinity for arginine and lysine, and was saturable and inhibited by mercurial compounds and other inhibitors of mitochondrial carriers to various degrees. The main physiological role of SLC25A29 is to import basic amino acids into mitochondria for mitochondrial protein synthesis and amino acid degradation.     

The Ecological Significance of Mycosporine‐Like Amino Acids in Algae

The links among mycosporine‐like amino acids (MAAs), algal physiological ecology, and the environment may be viewed from two sides. The UV‐absorption spectra of MAAs make them prime candidates for UV‐screening compounds, and indeed, numerous studies have shown that a high concentration of MAAs is correlated with various degrees of protection of cellular processes from UV damage. Thus, it might be said that the structure of terrestrial, eulittoral and sublittoral algal communities depends in part on their ability to synthesize effective quantities of MAAs. Corollaries to this hypothesis might include the proposition that those species incapable of synthesizing MAAs will be excluded from UV‐containing environments, and that UV is necessary to trigger MAA synthesis. However, a number of studies have demonstrated that triggers of MAA synthesis need not include exposure to UV, but may include the relative availability of nitrogen, osmotic stress, or high levels of photosynthetically active radiation. Furthermore, the response to these factors may vary as a function of other environmental conditions. These issues will be discussed with respect to elucidating the master switch for MAA synthesis.     

Cultivation of microalgae in the solution from the desalting process of soy sauce waste treatment and utilization of the algal biomass for ethanol fermentation

The growth of four marine microalgae was examined in a medium prepared from the effluent from the desalting process of soy sauce waste. A strain of Dunaliella that showed abundant growth on soy sauce waste extract was selected, and optimum cultivation conditions were determined. The algal cells produced were disrupted, and saccharified with glucoamylase. The saccharified solution was fermented using Saccharomyces cerevisiae IAM 4140. Stoichiometric study revealed that 11mg of ethanol was produced from 1g (dry cell weight) of Dunaliella cells. This work indicates a new method for removing waste products of the soy sauce production industry.     

植物对氨基酸的吸收及其生理效应

<正> 在古代的农业系统中,所用的肥料都是有机肥,人们并不知道植物依赖于何种物质得以生长繁衍。自1840年,德国化学家李比希(J.U.Liebig)发表了《化学在农业和植物生理学上应用》一书后,人们对植物的营养有了新的认识,认识到矿物质对植物的重要性。李比希在书中指出:“土壤中矿物质是一切绿色植物唯一的养料。植物可以完全依赖于无机物质而生长发育。这一观点即为“植物的矿质营养学说”。该学说后来为生产实践所充分证明,并成为农业化学     

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.     

Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen-Glucose Deprivation or Excitatory Amino Acids

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen-glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor-mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S -α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.     

烧伤患者血浆和红细胞游离氨基酸变化及输注氨基酸对其变化的影响

动态测定烧伤患者血浆及红细胞内游离氨基酸的含量 ,探讨输入外源性氨基酸后对血及红细胞内游离氨基酸的影响。以日立 835— 5 0型氨基酸自动分析仪测定烧伤患者血浆及红细胞内游离氨基酸含量。结果发现烧伤患者血浆总游离氨基酸浓度从伤后到 2 1天均显著降低 (P <0 .0 5～ 0 .0 1) ;赖、苯丙和苯丙 酪氨酸比值显著升高 (P <0 .0 5～ 0 .0 1) ;色、组、精、丙、甘、苏、脯和丝氨酸比值显著降低 (P <0 .0 5～ 0 .0 1) ;缬、亮、异亮、酪、胱和支链氨基酸伤后早期降低。烧伤患者红细胞内总游离氨基酸含量不同程度降低 ,其中 1、3、7天降低显著 (P <0 .0 5～ 0 .0 1) ;红细胞内苯丙和苯丙 酪氨酸比值未见显著性升高 ;色、蛋、精、脯氨酸含量很低或基本未测出。输注复合氨基酸注射液后未能显著改善患者血及红细胞内游离氨基酸含量。结果提示烧伤患者红细胞内游离氨基酸含量的变化趋势与血浆游离氨基酸变化趋势基本一致 ;烧伤后红细胞内苯丙氨酸及苯丙 酪氨酸比值有别于血浆变化。本研究条件下补充外源性氨基酸未能显著改变烧伤患者血浆及红细胞内游离氨基酸的含量     

The Effect of Nitrogen Nutrition on Cytokinin Activity and Free Amino Acids inBetula pendulaRoth, andAcer pseudoplatanusL.

A comparison was made of the levels of extractable cytokinin-likecompounds and free nitrate in the tissues of Betula pendulaand Acer pseudoplatanus at two levels of nitrogen nutrition.In further studies comparisons were made of the levels of cytokinin-likecompounds and free amino acids extracted from the tissues ofB. pendula supplied with nitrogen as ammonium sulphate, ammoniumnitrate, or calcium nitrate. These results were then relatedto growth differences between the treatments. B. pendula supplied with a low level of nitrogen as ammoniumnitrate continued to make slow growth whereas A. pseudoplatanusformed terminal resting buds after 2–3 weeks, furthergrowth being arrested. The low levels of extractable cytokinin-likecompounds found in the leaves of B. pendula receiving a lownitrogen supply were in contrast to the results from A. pseudoplatanus,where only a small reduction was found in response to low nitrogensupply. Analysis of the plant tissue showed that free nitrateand amino acids were depleted to a greater extent from B. pendulathan from A. pseudoplatanus under conditions of low nitrogensupply. It is suggested that the ability of B. pendula to continueactive growth with low nitrogen supply may be related to thereadiness with which nitrogen can be mobilized and re-circulatedin the plant, a process including sequential leaf senescence. Contrasting growth habits were seen in B. pendula in ammoniumnitrate- and ammonium sulphate-fed plants. Lateral shoot growthin ammonium sulphate-fed plants was completely inhibited andno detectable cytokinin activity was found in tissue extracts,whereas ammonium nitrate-fed plants grew rapidly, and showedconsiderable development of laterals. Changes in the levelsof individual amino acids show that the form in which nitrogenis taken up by plants affects the subsequent pathways of metabolism.