Novel Tryptophan Metabolism by a Potential Gene Cluster That Is Widely Distributed among Actinomycetes

The characterization of potential gene clusters is a promising strategy for the identification of novel natural products and the expansion of structural diversity. However, there are often difficulties in identifying potential metabolites because their biosynthetic genes are either silenced or expressed only at a low level. Here, we report the identification of a novel metabolite that is synthesized by a potential gene cluster containing an indole prenyltransferase gene (SCO7467) and a flavin-dependent monooxygenase (FMO) gene (SCO7468), which were mined from the genome of Streptomyces coelicolor A3(2). We introduced these two genes into the closely related Streptomyces lividans TK23 and analyzed the culture broths of the transformants. This process allowed us to identify a novel metabolite, 5-dimethylallylindole-3-acetonitrile (5-DMAIAN) that was overproduced in the transformant. Biochemical characterization of the recombinant SCO7467 and SCO7468 demonstrated the novel l-tryptophan metabolism leading to 5-DMAIAN. SCO7467 catalyzes the prenylation of l-tryptophan to form 5-dimethylallyl-l-tryptophan (5-DMAT). This enzyme is the first actinomycetes prenyltransferase known to catalyze the addition of a dimethylallyl group to the C-5 of tryptophan. SCO7468 then catalyzes the conversion of 5-DMAT into 5-dimethylallylindole-3-acetaldoxime (5-DMAIAOx). An aldoxime-forming reaction catalyzed by the FMO enzyme was also identified for the first time in this study. Finally, dehydration of 5-DMAIAOx presumably occurs to yield 5-DMAIAN. This study provides insight into the biosynthesis of prenylated indoles that have been purified from actinomycetes.     

High Doses of Vitamin B6 in the Rat Are Associated with Inhibition of Hepatic Tryptophan Metabolism and Increased Uptake of Tryptophan into the Brain

Abstract: Acute administration of vitamin B₆ to rats (10 mg/kg body weight) led to reduced urinary excretion of N ¹-methyl nicotinamide and methyl pyridone carboxamide, indicating inhibition of the oxidative metabolism of tryptophan. There was a considerable reduction in the production of ¹⁴CO₂ from [ ring -2-¹⁴C]tryptophan, and a significant inhibition of hepatic tryptophan oxygenase when measured in liver homogenates, together with an increase in the concentration of tryptophan in plasma. There was an increase in both the concentration of tryptophan in the brain and the uptake into the brain of peripherally administered [³H]tryptophan, accompanied by a small increase in the rate of synthesis of 5-hydroxy-tryptamine in the brain. It is suggested that this increase in the uptake of tryptophan into the brain following a relatively large dose of vitamin B₆ may explain the beneficial action of the vitamin in some cases of depressive illness.     

Monitoring the Effect of a Tryptophan Load on Brain Indole Metabolism in Freely Moving Rats by Simultaneous Cerebrospinal Fluid Sampling and Brain Dialysis

Rats were given L-tryptophan, 50 mg/kg i.p., and its concentration in the CNS was monitored in individual freely moving animals using repeated sampling of cisternal CSF and concurrent striatal dialysis. The 5-hydroxytryptamine metabolite 5-hydroxyindoleacetic acid (5-HIAA) was also measured. Results were compared with changes of central tryptophan and 5-HIAA concentrations in brains of rats killed at various times after administration of L-tryptophan, 50 mg/kg i.p. Tryptophan changes in CSF were proportionate to those in whole brain and followed essentially identical time courses. Results for the striatal dialysate and whole striatum also paralleled each other. Similarly, results for 5-HIAA showed proportionality between CSF and brain and between dialysate and striatum. The data obtained were used to determine pharmacokinetic data for individual rats, i.e., areas under curves for both tryptophan and 5-HIAA and half-lives for the decline of tryptophan. Kinetic parameters varied considerably from rat to rat. However, mean half-lives for tryptophan in CSF, brain, dialysate, and striatum were all comparable. Results in general show the value of repeated CSF sampling and intracerebral dialysis for concurrent monitoring of changes of indole metabolism in the whole brain and a specific brain region, respectively. The methods should be suitable for the continuous monitoring of changes of central transmitter metabolism in parallel with observation of behavior following environmental or dietary changes or drug administration. They also should be of use in the investigation of drug kinetics in the CNS.     

Expression and Deletion Mutagenesis of Tryptophan Hydroxylase Fusion Proteins: Delineation of the Enzyme Catalytic Core

Abstract: cDNAs encoding the full-length sequence for tryptophan hydroxylase, and deletion mutants consisting of the regulatory (amino acids 1–98) or catalytic (amino acids 99–444) domains of the enzyme, were cloned and expressed as glutathione S -transferase fusion proteins in E. coli . The recombinant fusion proteins could be purified to near homogeneity within minutes by affinity chromatography on glutathione-agarose. The full-length enzyme and the catalytic core expressed very high levels of tryptophan hydroxylase activity. The regulatory domain was devoid of activity. The full-length enzyme and the catalytic core, while adsorbed to glutathione-agarose beads, obeyed Michaelis-Menten kinetics, and the kinetic properties of each recombinant enzyme for cofactor and substrate compared very closely to native, brain tryptophan hydroxylase. Both active forms of the glutathione S -transferase-tryptophan hydroxylase fusion proteins had strict requirements for ferrous iron in catalysis and expressed much higher levels of activity ( V _max) than the brain enzyme. Analysis of full-length tryptophan hydroxylase and the catalytic core by molecular sieve chromatography under nondenaturing conditions revealed that each fusion protein behaved as a tetrameric species. These results indicate that a truncated tryptophan hydroxylase, consisting of amino acids 99–444 of the full-length enzyme, contains the sequence motifs needed for subunit assembly. Both wild-type tryptophan hydroxylase and the catalytic core are expressed as apoenzymes which are converted to holoenzymes by exogenous iron. The tryptophan hydroxylase catalytic core is also as active as the full-length enzyme, suggesting the possibility that the regulatory domain exerts a suppressive effect on the catalytic core of tryptophan hydroxylase.     

Exogenous Tryptophan Increases Synthesis, Storage, and Intraneuronal Metabolism of 5-Hydroxytryptamine in the Rat Hypothalamus

The effects of tryptophan administration on neurochemical estimates of synthesis [5-hydroxytryptophan (5-HTP) accumulation following administration of a decarboxylase inhibitor], storage [5-hydroxytryptamine (5-HT) concentrations], and metabolism [5-hydroxyindoleacetic acid (5-HIAA) concentrations] of 5-HT in selected regions of the hypothalamus were determined using HPLC coupled to an electrochemical detector. Tryptophan methyl ester HCl (30-300 mg/kg i.p.) produced a dose-dependent increase in the rate of 5-HTP accumulation throughout the hypothalamus but had no effect on the rate of accumulation of 3,4-dihydroxyphenylalanine. Peak 5-HTP levels were attained by 30 min following administration of tryptophan (100 mg/kg i.p.) and were maintained for an additional 60 min. Tryptophan also produced concomitant dose-dependent increases in 5-HT and 5-HIAA concentrations in these same regions without changes in the 5-HIAA/5-HT ratio. These results indicate that exogenous tryptophan administration selectively increases the synthesis, storage, and metabolism of 5-HT in the hypothalamus without altering the synthesis of catecholamines. Inhibition of 5-HT uptake with chlorimipramine or fluoxetine produced modest (10-40%) reductions in 5-HIAA concentrations throughout the hypothalamus, revealing that only a minor portion of 5-HIAA is derived from released and recaptured 5-HT, whereas the major portion of this metabolite reflects intraneuronal metabolism of unreleased 5-HT. In both chlorimipramine- and fluoxetine-treated rats, 5-HIAA concentrations were significantly increased by tryptophan administration, indicating that the increase in synthesis of 5-HT following precursor loading is accompanied by an increase in the intraneuronal metabolism of 5-HT.     

Oxindole, a Sedative Tryptophan Metabolite, Accumulates in Blood and Brain of Rats with Acute Hepatic Failure

Abstract: Rats treated with oxindole (10–100 mg/kg i.p.), a putative tryptophan metabolite, showed decreased spontaneous locomotor activity, loss of the righting reflex, hypotension, and reversible coma. Brain oxindole levels were 0.05 ± 0.01 nmol/g in controls and increased to 8.1 ± 1.7 or 103 ± 15 nmol/g after its administration at doses of 10 or 100 mg/kg i.p., respectively. To study the role that oxindole plays in the neurological symptoms associated with acute liver failure, we measured the changes of its concentration in the brain after massive liver damage, and we investigated the possible metabolic pathways leading to its synthesis. Rats treated with either thioacetamide (0.2 and 0.4 g/kg i.p., twice) or galactosamine (1 and 2 g/kg i.p.) showed acute liver failure and a large increase in blood or brain oxindole concentrations (from 0.05 ± 0.01 nmol/g in brains of controls to 1.8 ± 0.3 nmol/g in brains of thioacetamide-treated animals). Administration of tryptophan (300–1,000 mg/kg p.o.) caused a twofold increase, whereas administration of indole (10–100 mg/kg p.o.) caused a 200-fold increase, of oxindole content in liver, blood, and brain, thus suggesting that indole formation from tryptophan is a limiting step in oxindole synthesis. Oral administration of neomycin, a broad-spectrum, locally acting antibiotic agent able to reduce intestinal flora, significantly decreased brain oxindole content. Taken together, our data show that oxindole is a neurodepressant tryptophan metabolite and suggest that it may play a significant role in the neurological symptoms associated with acute liver impairment.     

Circadian Variations in the Activity of Tyrosine Hydroxylase, Tyrosine Aminotransferase, and Tryptophan Hydroxylase: Relationship to Catecholamine Metabolism

Abstract— Circadian variations in the activity of tyrosine hydroxylase, tyrosine aminotransferase, and tryptophan hydroxylase were observed in the rat brain stem. Tyrosine hydroxylase exhibited a bimodal pattern with peaks occurring during both the light and dark phases of the circadian cycle. Tyrosine aminotransferase had one daily peak of activity occurring late in the light phase, whereas tryptophan hydroxylase activity was maximal late in the dark phase. Circadian fluctuations in tyrosine hydroxylase activity did not correlate well with circadian variations in the turnover rates of norepinephrine or dopamine nor with levels of these catecholamines. This supports the idea that although tyrosine hydroxylase is the rate-limiting enzyme in the synthesis of catecholamines, other factors must also be involved in the in vivo regulation of this process. Administration of α -methyl- p -tyrosine (AMT) methyl ester HC1 (100 mg/kg) had no effect on the activity of tryptophan hydroxylase, but effectively eliminated the peak of tyrosine hydroxylase activity that occurred during the light phase. AMT also lowered levels of tyrosine aminotransferase, but only at times near the daily light to dark transition. These chronotypic effects of AMT emphasize the importance of "time of day" as a factor that must be taken into account in evaluating the biochemical as well as the pharmacological and toxicological effects of drugs.     

Mesenteric Organ Production, Hepatic Metabolism, and Renal Elimination of Norepinephrine and Its Metabolites in Humans

Abstract: This study used regional differences in plasma concentrations of norepinephrine and its metabolites to examine how production of the transmitter by sympathetic nerves, in particular, those innervating mesenteric organs, is integrated with metabolism by the liver and elimination by the kidneys. Higher concentrations of norepinephrine, its glycol metabolites 3,4-dihydroxyphenylglycol and 3-methoxy-4-hydroxyphenylglycol and their sulfate conjugates in portal venous than arterial plasma indicate substantial production of norepinephrine by mesenteric organs (15.5 nmol/min). Much lower concentrations of norepinephrine and its glycol metabolites in plasma leaving than entering the liver indicate their efficient hepatic removal (20 nmol/min). Higher concentrations of vanillylmandelic acid in the hepatic outflow than inflow indicate that this metabolic end product is produced largely from the norepinephrine and glycol metabolites removed by the liver. Renal elimination of vanillylmandelic acid (18–20 nmol/min), produced mainly by the liver (17 nmol/min), and of 3-methoxy-4-hydroxyphenylglycol sulfate (7–9 nmol/min), produced largely by mesenteric organs (7 nmol/min), comprised 86–91% of the total renal elimination of norepinephrine metabolites. The results show that mesenteric organs produce about one-half of the norepinephrine formed in the body. The liver removes substantial amounts of circulating norepinephrine and its glycol metabolites and converts these compounds to vanillylmandelic acid, which is then eliminated from the body by the kidneys. The sulfate conjugates are also metabolic end products eliminated by the kidneys. However, these metabolites are produced by extrahepatic tissues, in particular, mesenteric organs, which represent a significant source of sulfate-conjugated norepinephrine and 3,4-dihydroxyphenylglycol, and the main source of sulfate-conjugated 3-methoxy-4-hydroxyphenylglycol.     

抑癌基因p53 在肝癌组织中的异常表达及临床意义

目的:探讨抑癌基因p53在肝癌组织中表达及临床意义,为肝癌的治疗提供新的思路。方法:回顾性分析了2006年1月～2009年8月收治的40例肝癌患者的临床资料,采用免疫组织化学法测定肝癌组织、癌旁组织和正常组织中抑癌基因p53的表达,并分析不同肿瘤分化程度患者p53的表达。结果:p53阳性表达率在在肝癌组、癌旁组分别为47.5%和2.5%,在正常组未检测到p53阳性表达,三组之间比较有显著性差异(x2=6.515,P=0.024);高分化癌P53蛋白阳性表达率低,中低分化癌P53蛋白阳性表达率高,组比较均有显著性差异(P<0.05),中、低分化组之间比较差异无统计学意义(P>0.05)。结论:在肝癌组织中存在p53基因的高表达,其阳性表达与肿瘤分化程度有关。     

电穿孔下离子型药物透皮扩散的一类数学模型及其建模方法论

在分子型模型的基础上,导出了离子型药物经皮渗透过程的一类数学模型,并用计算机模拟了其特性、而且针对分子型和离子型模型中,待估参数相对于实验样本点而言过多,同时需处理大批在不同实验控制条件下的数据资料的情形,讨论了其建模方法论问题。     

MRS脑肿瘤分型诊断模型研究

基于对核磁波谱(MR Spectroscopy)的特征变量提取,利用模糊聚类算法来构建脑肿瘤分型诊断模型,实验结果表明该模型能准确对神经胶质细胞瘤分级诊断,为进一步实现更复杂的脑肿瘤诊断系统模型提供方法。     

Metabolic Analysis of Wild-type Escherichia coli and a Pyruvate Dehydrogenase Complex (PDHC)-deficient Derivative Reveals the Role of PDHC in the Fermentative Metabolism of Glucose

Pyruvate is located at a metabolic junction of assimilatory and dissimilatory pathways and represents a switch point between respiratory and fermentative metabolism. In Escherichia coli, the pyruvate dehydrogenase complex (PDHC) and pyruvate formate-lyase are considered the primary routes of pyruvate conversion to acetyl-CoA for aerobic respiration and anaerobic fermentation, respectively. During glucose fermentation, the in vivo activity of PDHC has been reported as either very low or undetectable, and the role of this enzyme remains unknown. In this study, a comprehensive characterization of wild-type E. coli MG1655 and a PDHC-deficient derivative (Pdh) led to the identification of the role of PDHC in the anaerobic fermentation of glucose. The metabolism of these strains was investigated by using a mixture of ¹³C-labeled and -unlabeled glucose followed by the analysis of the labeling pattern in protein-bound amino acids via two-dimensional ¹³C,¹H NMR spectroscopy. Metabolite balancing, biosynthetic ¹³C labeling of proteinogenic amino acids, and isotopomer balancing all indicated a large increase in the flux of the oxidative branch of the pentose phosphate pathway (ox-PPP) in response to the PDHC deficiency. Because both ox-PPP and PDHC generate CO₂ and the calculated CO₂ evolution rate was significantly reduced in Pdh, it was hypothesized that the role of PDHC is to provide CO₂ for cell growth. The similarly negative impact of either PDHC or ox-PPP deficiencies, and an even more pronounced impairment of cell growth in a strain lacking both ox-PPP and PDHC, provided further support for this hypothesis. The three strains exhibited similar phenotypes in the presence of an external source of CO₂, thus confirming the role of PDHC. Activation of formate hydrogen-lyase (which converts formate to CO₂ and H₂) rendered the PDHC deficiency silent, but its negative impact reappeared in a strain lacking both PDHC and formate hydrogen-lyase. A stoichiometric analysis of CO₂ generation via PDHC and ox-PPP revealed that the PDHC route is more carbon- and energy-efficient, in agreement with its beneficial role in cell growth.     

Motor Activity Increases Tryptophan, 5-Hydroxyindoleacetic Acid, and Homovanillic Acid in Ventricular Cerebrospinal Fluid of the Conscious Rat

An investigation was made into the effects of running (1 h at 20 m/min) on central serotonergic and dopaminergic metabolism in trained rats. Methodology involved continuous withdrawal of cerebrospinal fluid (CSF) from the third ventricle of conscious rats and measurements of tryptophan (TRP), 5-hydroxyindoleacetic acid (5-HIAA), and homovanillic acid (HVA) levels during a 2 h post-exercise period. All three compounds were increased during the hour following exercise and returned to their basal values within an hour later. CSF flow rate was stable when metabolite levels were elevated. Brain determinations indicated that CSF metabolite variations only qualitatively paralleled brain changes. Indeed, post-exercise TRP, 5-HIAA, and HVA levels were increased to a greater extent in brain when compared to CSF. It is suggested that increased serotonergic and dopaminergic metabolism, caused by motor activity, may be involved in the behavioral effects of exercise.     

猪JARID1C基因的cDNA克隆、生物信息学及组织表达谱分析

JARID1C是高度保守的ARID蛋白家族的成员,该家族的蛋白参与并引起一系列生物学效应,如染色质重塑、细胞增殖与分裂、个体发育以及基因转录调控。JARID1C在人脑中表达丰富,对脑的发育和维持正常功能具有重要作用,突变可引起智力迟钝。本研究采用电子克隆（insilicocloning）的方法并结合5′末端快速扩增技术（RACE）,从猪卵巢中克隆到JARID1C的全长cDNA序列（GenBank登录号：EF139241）。猪JARID1C基因的cDNA全长5,908bp,包括4,551bp的开放阅读框（ORF）、522bp的5′非翻译区（5′UTR）和835bp的3′非翻译区（3′UTR）,polyA加尾信号序列AATAAA位于5,881bp和5,886bp之间。生物信息学分析揭示JARID1C蛋白含有1517个氨基酸残基,定位于细胞核中,该蛋白含有5个保守的结构域：JmjN结构域、ARID结构域、JmjC结构域、C5HC2锌指结构域和PHD锌指结构域。应用ClusterW程序分别对猪、狗、小鼠、大鼠、人和猿的JARID1C核苷酸序列和氨基酸序列进行多重序列比对,发现猪的JARID1C与其他哺乳动物具有很高的相似性。借助Mega3.1软件,采用N-J算法构建JARID1亚家族蛋白的系统进化树,揭示不同物种的进化关系。应用实时荧光定量PCR技术分析该基因在不同组织的表达差异,结果表明该基因在各组织均不同程度地表达,其中在肺和骨骼肌表达水平最低,而在脑和性腺表达水平最高。