Mechanism of reaction of 3-hydroxyanthranilic acid with molecular oxygen

The autoxidation of the tryptophan metabolite, 3-hydroxyanthranilic acid, at pH 7 gives rise to a p-quinone dimer and cinnabarinic acid. A novel dimer formed by radical-radical coupling of 3-hydroxyanthranilic acid is also produced. Labelling studies have shown that the C-2 oxygen in the p-quinone dimer is derived from molecular oxygen. A product versus time study of this reaction has revealed that, in the absence of catalase, cinnabarinic acid is formed but undergoes decomposition by hydrogen peroxide. At pH 7, in the presence of catalase, both the p-quinone dimer and cinnabarinic acid are formed at approximately the same rate and this rate of formation increases with increasing pH. Inclusion of superoxide dismutase was found to increase the rate of formation of cinnabarinic acid, suggesting that superoxide ions may also cause decomposition of cinnabarinic acid. This was confirmed by treating cinnabarinic acid with superoxide. A mechanism involving a common anthranilyl radical intermediate is proposed to account for the formation of the different oxidation products.     

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.     

Involvement of oxidoreductive reactions of intracellular haemoglobin in the metabolism of 3-hydroxyanthranilic acid in human erythrocytes.

3-Hydroxyanthranilic acid, a metabolite of tryptophan, was rapidly metabolized by human erythrocytes. The final product was determined to be cinnabarinic acid as detected by spectrophotometry, paper chromatography and t.l.c. The formation of cinnabarinic acid from 3-hydroxyanthranilic acid in the cells was markedly inhibited by CO when intracellular haemoglobin was in a ferrous state, and by cyanide when it was in a ferric state. Ferrous haemoglobin in erythrocytes was oxidized to (alpha 3+ beta 2+)2, (alpha 2+ beta 3+)2 and (alpha 3+ beta 3+)2 by 3-hydroxyanthranilic acid, and the oxidation rates were very high, like those of cinnabarinic acid formation, suggesting that the metabolism of 3-hydroxyanthranilic acid is coupled with oxidoreductive reactions of intracellular haemoglobin. This view was further confirmed by the findings that 3-hydroxyanthranilic acid was metabolized by ferrous or ferric haemoglobin and that ferrous and ferric haemoglobins were oxidized and reduced by the compound respectively. The significance of the metabolism of 3-hydroxyanthranilic acid and the oxidoreductive reactions of haemoglobin with this compound may be associated with the pathological conditions with increased 3-hydroxyanthranilic acid levels in the blood of diabetic subjects.     

Novel Interaction between Laccase and Cellobiose Dehydrogenase during Pigment Synthesis in the White Rot Fungus Pycnoporus cinnabarinus

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.     

家蚕中的5-氧脯氨酸酶及保幼激素类似物对它的影响

本工作首次在家蚕Bombyx mori的马氏管、中肠、丝腺及脂肪体等组织中测到了γ-谷氨酰循环中一个关键酶——5-L-氧脯氨酸酶的活力.该酶以马氏管中活力最高,在蚕的中肠、血淋巴中均存在有游离的5-氧脯氨酸.观察了保幼激素类似物(JHA)处理后,家蚕中肠、丝腺和脂肪体中5-L-氧脯氨酸酶活力的变化,同时观察了血淋巴中5-氧脯氨酸含量的变化.对该酶及γ-谷氨酰循环在蚕体中氨基酸转运上的可能作用进行了讨论.     

Cloning and partial characterization of a gene in Bombyx mori homologous to a human adiponectin receptor

In this study, we report the cloning and characteristics of an adiponectin-like receptor gene from Bombyx mori (BmAdipoR) with highly conserved deduced amino-acid sequences and similar structure to the human adiponectin receptor (AdipoR). Structural analysis of the translated cDNA suggested it encoded a membrane protein with seven transmembrane domains. BmAdipoR was found to be expressed in multiple tissues and highly expressed in Malpighian tubules, fat body and testis. BmNPV (Bombyx mori nucleopolyhedrovirus) bacmid system combined with confocal microscopy revealed that BmAdipoR was targeted to the cell membrane. We also found that infection with BmNPV did not have an effect on BmAdipoR mRNA quantity in the midgut of susceptible Bombyx mori strain (306) at 48 h, but BmAdipoR mRNA quantity increased significantly at 72 h. We concluded that BmAdipoR gene was a membrane protein ubiquitously expressed in Bombyx mori tissues and that its expression was altered by treating with BmNPV.     

Effects of mitochondria and o-methoxybenzoylalanine on 3-hydroxyanthranilic acid dioxygenase activity and quinolinic acid synthesis

The use of o-methoxybenzoylalanine, a selective kynureninase inhibitor, has been proposed with the aim of reducing brain synthesis of quinolinic acid, an excitotoxic tryptophan metabolite. In liver homogenates, however, this compound caused unexpected accumulation of 3-hydroxyanthranilic acid, the product of kynureninase activity and the precursor of quinolinic acid. To explain this observation, we investigated the interaction(s) of o-methoxybenzoylalanine with 3-hydroxyanthranilic acid dioxygenase, the enzyme responsible for quinolinic acid formation. When the purified enzyme or partially purified cytosol preparations were used, o-methoxybenzoylalanine did not affect 3-hydroxyanthranilic acid dioxygenase activity. However, a significant reduction of this enzymatic activity did occur when o-methoxybenzoylalanine was tested in the presence of mitochondria. It is interesting that addition of purified mitochondria to 3-hydroxyanthranilic acid dioxygenase preparations reduced the enzymatic activity and the synthesis of quinolinic acid. In vivo, administration of o-methoxybenzoylalanine significantly reduced quinolinic acid synthesis and content in both blood and brain of mice. Our results suggest that mitochondrial protein(s) interact(s) with soluble 3-hydroxyanthranilic acid dioxygenase and cause(s) modifications in the enzyme resulting in a decrease in its activity. These modifications also allow the enzyme to interact with o-methoxybenzoylalanine, thus leading to a further reduction in quinolinic acid synthesis.     

Utilization of free fatty acids by starved and pregnant sheep

Rat-liver cinnabarinate synthase (3-hydroxyanthranilic acid-oxygen oxido-reductase) was partially purified. Stoicheiometric studies indicated the consumption of 3 atoms of oxygen/molecule of cinnabarinic acid formed. There was an initial lag in enzyme activity. The reaction had an optimum pH about 7.2 and an optimum temperature of 37 degrees . The enzyme was highly specific for 3-hydroxyanthranilic acid. The system showed an absolute requirement for Mn(2+) ions. Several bivalent metal ions and metal-chelating agents inhibited the reaction. Thiol inhibitors had no effect on enzyme activity, but reducing agents such as ascorbic acid were potent inhibitors. There was no requirement for any cofactor other than Mn(2+) ions. The probable significance of the reaction in mammals is discussed.     

Superoxide dismutase enhances the formation of hydroxyl radicals in the reaction of 3-hydroxyanthranilic acid with molecular oxygen.

Superoxide dismutase (SOD) enhanced the formation of hydroxyl radicals, which were detected by using the e.s.r. spin-trapping technique, in a reaction mixture containing 3-hydroxyanthranilic acid (or p-aminophenol), Fe3+ ions, EDTA and potassium phosphate buffer, pH 7.4. The hydroxyl-radical formation enhanced by SOD was inhibited by catalase and desferrioxamine, and stimulated by EDTA and diethylenetriaminepenta-acetic acid, suggesting that both hydrogen peroxide and iron ions participate in the reaction. The hydroxyl-radical formation enhanced by SOD may be considered to proceed via the following steps. First, 3-hydroxyanthranilic acid is spontaneously auto-oxidized in a process that requires molecular oxygen and yields superoxide anions and anthranilyl radicals. This reaction seems to be reversible. Secondly, the superoxide anions formed in the first step are dismuted by SOD to generate hydrogen peroxide and molecular oxygen, and hence the equilibrium in the first step is displaced in favour of the formation of superoxide anions. Thirdly, hydroxyl radicals are generated from hydrogen peroxide through the Fenton reaction. In this Fenton reaction Fe2+ ions are available since Fe3+ ions are readily reduced by 3-hydroxyanthranilic acid. The superoxide anions do not seem to participate in the reduction of Fe3+ ions, since superoxide anions are rapidly dismuted by SOD present in the reaction mixture.     

Oxidation of 3-hydroxyanthranilic acid to the phenoxazinone cinnabarinic acid by peroxyl radicals and by compound I of peroxidases or catalase.

Since 3-hydroxyanthranilic acid (3HAA), an oxidation product of tryptophan metabolism, is a powerful radical scavenger [Christen, S., Peterhans, E., & Stocker, R. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2506], its reaction with peroxyl radicals was investigated further. Exposure to aqueous peroxyl radicals generated at constant rate under air from the thermolabile radical initiator 2,2'-azobis[2-amid-inopropane] hydrochloride (AAPH) resulted in rapid consumption of 3HAA with initial accumulation of its cyclic dimer, cinnabarinic acid (CA). The initial rate of formation of the phenoxazinone CA accounted for approximately 75% of the initial rate of oxidation of 3HAA, taking into account that 2 mol of 3HAA are required to form 1 mol of CA. Consumption of 3HAA under anaerobic conditions (where alkyl radicals are produced from AAPH) was considerably slower and did not result in detectable formation of CA. Addition of superoxide dismutase enhanced autoxidation of 3HAA as well as the initial rates of peroxyl radical-induced oxidation of 3HAA and formation of CA by approximately 40-50%, whereas inclusion of xanthine/xanthine oxidase decreased the rate of oxidation of 3HAA by approximately 50% and inhibited formation of CA almost completely, suggesting that superoxide anion radical (O2.-) was formed and reacted with reaction intermediate(s) to curtail formation of CA. Formation of CA was also observed when 3HAA was added to performed compound I of horseradish peroxidase (HRPO) or catalytic amounts of either HRPO, myeloperoxidase, or bovine liver catalase together with glucose/glucose oxidase.(ABSTRACT TRUNCATED AT 250 WORDS)     

家蚕Wnt信号通路下游关键基因Pangolin转录剪接体X3的克隆和分析

【目的】克隆家蚕Bombyx mori Wnt信号通路下游关键基因Pangolin isoforms A/H/I/S转录剪接体X3 (Pangolin X3),分析其序列和表达特征。【方法】从NCBI数据库检索家蚕Pangolin X3,根据其编码序列(coding sequence, CDS)设计引物,利用PCR从家蚕幼虫中肠和血淋巴中进行克隆并测序验证。利用SilkDB 3.0, SMART,多序列比对和系统发育树分析Pangolin X3的序列特征。利用qRT-PCR分析Pangolin X3在家蚕5龄第3天幼虫不同组织(头、血淋巴、体壁、性腺、中肠、前部丝腺、中部丝腺、后部丝腺、脂肪体和马氏管)中的相对表达水平。【结果】从家蚕幼虫中肠和血淋巴克隆了Pangolin X3(GenBank登录号：XM＿038020921)的CDS,其开放阅读框长1 560 bp,编码519个氨基酸残基,预测分子量为55.86 kD,预测等电点为7.53。Pangolin X3蛋白含有保守的β-catenin结合位点和HMG结构域,其氨基酸序列在不同的昆虫中比较保守,特别是与DNA结合的HMG结构域...     

Post-eclosion diuresis in a flightless insect, the silkmoth Bombyx mori

Abstract. Although the silkmoth, Bombyx mori L., has lost the ability to fly, it has retained a post-eclosion diuresis. In moths removed from their cocoons before eclosion, or in those which failed to spin cocoons as larvae, the weight loss due to diuresis was 14% of the eclosion body weight in males. Moths which used labial fluid to escape from their cocoons showed a correspondingly smaller diuresis (5%). Both urine and labial fluid had high potassium and low sodium concentrations. Unlike post-eclosion diuresis in butterflies, however, the urine was isosmotic to the haemolymph. In vitro preparations of B.mori Malpighian tubules were stimulated by cyclic AMP, B.mori brain extracts and Manduca sexta diuretic peptide (Mas-DP I).     

Synthesis of cinnabarinic acid by metabolically engineered Pseudomonas chlororaphis GP72

Cinnabarinic acid is a valuable phenoxazinone that has broad applications in the pharmaceutical, chemical, and dyeing industries. However, few studies have investigated the production of cinnabarinic acid or its derivatives using genetically engineered microorganisms. Herein, an efficient synthetic pathway of cinnabarinic acid was designed and constructed in Pseudomonas chlororaphis GP72 for the first tim, which was more straightforward and robust than the known eukaryotic biosynthetic pathways. First, we screened and identified trans-2,3-dihydro-3-hydroxyanthranilic acid (DHHA) dehydrogenases from Escherichia coli MG1655 (encoded by entA), Streptomyces sp. NRRL12068 (encoded by bomO) and Streptomyces chartreusis NRRL3882 (encoded by calB₃) based on the structural similarity of the substrate and product, and the DHHA dehydrogenase encoded by calB₃ was selected for the synthesis of cinnabarinic acid due to its high DHHA conversion rate. Subsequently, cinnabarinic acid was synthesized by the expression of the DHHA dehydrogenase CalB₃ and the phenoxazinone synthase CotA in the DHHA-producing strain P. chlororaphis GP72, resulting in a cinnabarinic acid titer of 20.3 mg/L at 48 hr. Further fermentation optimization by the addition of Cu²⁺, H₂O₂, and with adding glycerol increased cinnabarinic acid titer to 136.2 mg/L in shake flasks. The results indicate that P. chlororaphis GP72 may be engineered as a microbial cell factory to produce cinnabarinic acid or its derivatives from renewable bioresources.     

Enzymic conversion of 3-hydroxanthranilic acid into cinnabarinic acid by the nuclear fraction of rat liver

1. An enzyme solely localized in the nuclear fraction of rat liver was found to convert 3-hydroxyanthranilic acid into a red product that was isolated and crystallized from the reaction mixture. The product was identified as cinnabarinic acid (2-amino-3-oxo-3H-phenoxazine-1,9-dicarboxylic acid) by comparing its properties with synthetic cinnabarinic acid. 2. The enzyme had optimum pH at 7·2. Heavy-metal ions like Ag⁺, Hg²⁺, MoO₄²⁻, Fe²⁺ and Cu²⁺ were inhibitory; Mn²⁺ activated the reaction to a considerable extent. 3. The reaction was inhibited by mercaptoethanol, GSH and cysteine, and activated by p-hydroxymercuribenzoate and sodium arsenite, which may suggest the involvement of disulphide groups in the reaction.     

家蚕生存素基因在BmN4细胞有丝分裂中的功能分析

【目的】阐明生存素基因在家蚕Bombyx mori BmN4细胞有丝分裂中的功能。【方法】qRT PCR分析家蚕生存素基因BmSurvivin在家蚕5龄第3天幼虫不同组织(丝腺、中肠、马氏管、精巢、卵巢、脂肪体、皮细胞层和表皮)中的表达量;构建pIZT/V5-His-BmSurvivin-GFP (BG)融合载体并转染BmN4细胞,以免疫荧光标记检测BmSurvivin和第10位丝氨酸(Ser10)磷酸化的组蛋白H3(H3Ser10ph)在BmN4细胞有丝分裂分裂不同时期的定位。【结果】BmSurvivin在家蚕5龄第3天幼虫马氏管中表达量最高,其次是在丝腺和中肠中。成功构建pIZT/V5-His-BmSurvivin-GFP载体。免疫荧光结果显示,在BmN4细胞间期核中可以看到明显的GFP信号,涵盖了细胞核和细胞质区域,指示BmSurvivin的定位;随着细胞进入分裂期,GFP信号指示BmSurvivin与染色质共定位,待细胞形成明显的双取向时在纺锤体区域也可以看到GFP信号;后期随着姐妹染色单体分开,GFP信号指示BmSurvivin定位在染色质及胞质分裂区;末期随着两个新的子细胞形成GFP信号指示BmSurvivin仅定位在胞质分裂区。H3Ser10ph定位在BmN4细胞前中期染色质凝缩处,至中期信号最强,与整条染色体重合,后期信号消失。【结论】BmSurvivin与有丝分裂周期中染色质和纺锤体的动态变化相关。     

Cloning of human 3-hydroxyanthranilic acid dioxygenase in Escherichia coli: characterisation of the purified enzyme and its in vitro inhibition by Zn2+

3-hydroxyanthranilic acid oxygenase (3-HAO) catalyses the conversion of 3-hydroxyanthranilic acid to quinolinic acid. Because of the involvement of quinolinic acid in the initiation of neurodegenerative phenomena, we have cloned human 3-HAO in Escherichia coli, overexpressed and purified it with the aim of studying its enzymatic activity and for future structural studies. The recombinant human protein, obtained in E. coli, retains its enzymatic activity which can occur only in the presence of Fe(II); several other metals have been tested but in no case the formation of the product has been observed. On the contrary, two of the ions tested inhibit the catalytic reaction and one of them, Zn2+, could be of physiological relevance. A circular dichroism analysis has also been performed, showing that the secondary structure is mainly of the beta type, with a minority of alpha.     

吡哆醛激酶基因在家蚕体内的时空表达特征

【目的】了解家蚕Bombyx mori维生素B₆关键代谢酶吡哆醛激酶（pyridoxal kinase, PLK）在家蚕不同组织间的表达差异。【方法】原核表达家蚕重组PLK, 获得目的蛋白制备多克隆抗体, 利用Western blot方法对PLK在家蚕不同发育时期、 5龄第3天幼虫不同组织及5龄不同日龄幼虫表皮、 头部和马氏管中的表达进行分析; 并采用实时荧光定量PCR的方法对PLK基因在家蚕不同组织中的mRNA转录水平进行比较。【结果】PLK在家蚕5龄幼虫的表达水平最高, 在5龄第3天幼虫各组织中的表达由高到低依次为： 精巢、 马氏管、 表皮、 中肠、 头部、 卵巢、 脂肪体、 丝腺; 5龄期不同日龄幼虫表皮组织的表达差异最大, 头部和马氏管中均为前期表达量稍高于后期。在转录水平方面, 精巢的表达量最高, 其次是马氏管和头部。【结论】PLK在家蚕不同发育时期及组织中的差异表达进一步证实PLK在家蚕VB6代谢中的生物学功能的重要性。     

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.     

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.