Role of the uvr gene in the SOS function inducing activity of two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, in Escherichia coli K12

Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS-chromotest using PQ 37 (uvr A) and PQ 35 (uvr+) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 less than Trp-P-2 less than aflatoxin B1, at low concentrations (less than 125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr+ strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp-P-1 and Trp-P-2. At higher concentrations (125-500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration.     

Effect of glutathione and uridine-5'-diphosphoglucuronic acid on the mutagenicity of tryptophan pyrolysis products (Trp-P-1 and Trp-P-2) by rat-liver and -intestine S9 fraction

In the Ames test, after the addition of glutathione (GSH) or uridine-5'-diphosphoglucuronic acid (UD-PGA), we observed for Trp-P-1 an unchanged or a reduced mutagenicity by both the liver and intestine S9 fraction. For Trp-P-2, the same was true when we used the intestine S9 fraction. In the presence of liver S9 fraction, Trp-P-2 mutagenicity was also decreased by the addition of UDPGA but was increased by the addition of GSH. These results show that cofactors for glucuronide and GSH conjugation may alter the metabolic activation of Trp-P-1 and Trp-P-2 and consequently their mutagenicity.     

Analysis of human plasma as an exposure level monitor for carcinogenic tryptophan pyrolysis products

A high-performance liquid chromatography method for detecting 3-amino-1,4- dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in human plasma was developed. Plasma samples of 10 normal subjects were examined. Trp-P-1 and Trp-P-2, carcinogenic tryptophan pyrolysis products, were detected in all specimens, and the concentrations of Trp-P-1 and Trp-P-2 in plasma were 68.31 +/- 24.03 fmoles/ml (mean +/- S.D., n = 10) and 18.79 +/- 4.99 fmoles/ml, respectively. Our results suggest that plasma levels of carcinogenic tryptophan pyrolysis products may be useful indicators for estimating the exposure levels of the dietary carcinogens.     

Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.     

Carcinogenic tryptophan pyrolysis products in airborne particles and rain water

This is the first report that carcinogenic tryptophan pyrolysis products are present in airborne particles and rain water. The airborne particles were collected from August 1988 through October 1988 at 4 locations in Japan. The amounts of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2) in the air were 0.23 +/- 0.17 pg/m3 air (mean +/- SD, n = 18) and 0.16 +/- 0.15 pg/m3 air (n = 18), respectively. Moreover, these carcinogens were detected in rain water. These results indicate that Trp-P-1 and Trp-P-2 are ubiquitous environmental components.     

Oxidatively-modified and glycated proteins as candidate pro-inflammatory toxins in uremia and dialysis patients

End stage renal disease (ESRD) patients accumulate blood hallmarks of protein glycation and oxidation. It is now well established that these protein damage products may represent a heterogeneous class of uremic toxins with pro-inflammatory and pro-oxidant properties. These toxins could be directly involved in the pathogenesis of the inflammatory syndrome and vascular complications, which are mainly sustained by the uremic state and bioincompatibility of dialysis therapy. A key underlying event in the toxicity of these proteinaceous solutes has been identified in scavenger receptor-dependent recognition and elimination by inflammatory and endothelial cells, which once activated generate further and even more pronounced protein injuries by a self-feeding mechanism based on inflammation and oxidative stress-derived events. This review examines the literature and provides original information on the techniques for investigating proteinaceous pro-inflammatory toxins. We have also evaluated therapeutic - either pharmacological or dialytic - strategies proposed to alleviate the accumulation of these toxins and to constrain the inflammatory and oxidative burden of ESRD.     

Evaluation of the capacity of the liver for phenazone biotransformation in patients with uremia on peritoneal dialysis

This work aimed at establishing whether liver ability to biotransformation of drugs expressed by antipyrine kinetics is disturbed in peritoneally dialysed patients with end-stage renal failure. The investigations were carried out in 10 uraemic patients using the antipyrine test and comparing antipyrine kinetics with those obtained in 13 healthy individuals. At the time of investigations, standard clinical tests of liver function were normal and HBs antigen was absent in all patients. It was shown that peritoneally dialysed patients with end-stage renal failure had not significantly changed antipyrine elimination as compared with the group of healthy controls: t0.5 = 13.2 +/- 6.8 v. 11.8 +/- 8.1 h, plasma clearance = 50 +/- 30 v. 34 +/- 21 ml/min (x +/- SD). The obtained results indicate that antipyrine kinetics is within normal range in uraemic patients regularly dialysed suggesting cytochrome P-450 in microsomes not being markedly reduced.     

Controlled trial of enalapril in patients with chronic fluid overload undergoing dialysis

About one third of patients receiving dialysis for end stage renal failure have chronic fluid overload despite advice to restrict their oral fluid intake. To investigate the potential of an angiotensin converting enzyme inhibitor in reducing the urge to drink and consequent gain in weight, a double blind, placebo controlled crossover trial of enalapril was conducted in 25 patients receiving dialysis who had fluid overload. The trial comprised a baseline period of four weeks; two periods of treatment, each of four weeks, during which patients received either placebo or enalapril 5 mg twice each week; and a follow up period of four weeks. Five patients withdrew from the trial, one because of an adverse drug reaction to enalapril. A range of biochemical and behavioural variables was measured during the baseline period, at the completion of periods 1 and 2, and during follow up. These variables included gain in weight between dialysis sessions; blood pressure; plasma concentrations of sodium, angiotensin II, and vasopressin; plasma renin and angiotensin converting enzyme activities; osmolality; and estimations of thirst, intake of fluid, and control of drinking. Enalapril caused a significant reduction in gain in weight between dialysis sessions, thirst, and oral intake of fluid in parallel with significantly increased renin activity, significantly decreased angiotensin converting enzyme activity, and decreased concentrations of angiotensin II. Gain in weight and angiotensin converting enzyme activity returned to baseline values once patients stopped taking enalapril.These results suggest that enalapril may act on the renin-angiotensin system and reduce intake of fluid by inhibiting angiotensin converting enzyme.     

Phagocytosis and neutrophil bactericidal capacity in patients with uremia

The phagocytic activity and bactericidal capacity of polymorphonuclear neutrophils (PMN) were evaluated in patients with advanced chronic renal failure. The studies were made in patients undergoing hemodialysis, maintenance peritoneal dialysis as well as in nondialysed patients. Evaluations were carried out by using of the recently described fluorochrome microassay which enabled these parameters to be estimate independently. The phagocytic activity was seriously diminished in nondialysed patients, whereas it was similar to controls in those hemodialysed and undergoing peritoneal dialysis patients. In all evaluated groups of patients the bactericidal capacity was significantly reduced. The lowest values could always be observed in nondialysed patients. The decrease of bactericidal capacity was significantly more evident in patients undergoing peritoneal dialysis as compared with those hemodialysed. The obtained results confirm some previous reports suggesting the impairment of PMN function in uremic patients. This results in their increased susceptibility to infection. They also reveal the existence of a close relationship between the extent of observed dysfunctions and the management applied.     

Cytokines correlate with age in healthy volunteers, dialysis patients and kidney-transplant patients

T-cell functions are currently used as biomarkers for the pharmacodynamic monitoring of immunosuppressive drugs or as disease biomarkers of inflammation/sepsis and organ rejection. In order to evaluate co-factors potentially influencing the expression of the immunological biomarkers, we explored T-cell proliferation, T-cell activation (CD25 and CD71 expressions) and intra-lymphocyte cytokine production (interleukin (IL)-2 and tumor necrosis factor (TNF)-α) in healthy volunteers, dialysis patients and stable kidney-transplant patients treated with standard immunosuppressive therapy, i.e. tacrolimus, mycophenolic acid with or without steroids. Age was positively correlated with TNF-α expression in all three patient populations, and with IL-2 expression in healthy volunteers and kidney-transplant patients. Further age was correlated with inhibition of lymphocyte proliferation in healthy volunteers and with the T-cell activation marker CD25 in kidney-transplant patients. In healthy volunteers lymphocyte proliferation was higher in woman as compared to men. Other biomarkers of T-cell function were independent of the gender. In the kidney-transplant patient group a significantly lower expression of all biomarkers of T-cell functions compared to healthy volunteers and dialysis patients. In dialysis patients we found significant increased IL-2 expression compared to healthy volunteers, while the other T-cell functions were not significantly different. Further time on dialysis had no effect on the level of biomarker expression. In conclusion we found decreased T-cell functions in kidney-transplant patients compared to healthy volunteers and dialysis patients, increased IL-2 expression in dialysis patients compared to healthy volunteers and in all three populations we found a correlation of age and intra-T-lymphocyte TNF-α expression.     

鼻咽癌患者鼻咽灌洗液菌群检测及结果分析

目的 采用聚合酶链式反应－变性梯度凝胶电泳（PCR-DGGE）技术,通过观察鼻咽癌患者与健康对照者鼻咽灌洗液菌群的变化,初步探讨菌群异常在鼻咽癌发生中的作用。方法 以7例明确诊断且尚未治疗的鼻咽鳞癌患者和5例健康对照者为研究对象,提取鼻咽灌洗液DNA,观察两组研究对象菌群分布差异,进行相似性、多样性分析。结果 鼻咽癌患者与健康对照者之间鼻咽灌洗液的菌群分布存在差异,与健康对照者比较,咽颊炎链球菌和不动杆菌数量增加,但嗜热链球菌数量减少。结论 鼻咽癌患者鼻咽灌洗液菌群分布与健康对照者比较出现明显差异,可为鼻咽癌预防及治疗提供实验依据。     

Production of a novel tryptophan analog, beta-1-indazole-L-alanine with tryptophan synthase of Escherichia coli

The tryptophan synthase alpha 2 beta 2 complex from Escherichia coli has been found to catalyze the beta-replacement reaction of L-serine with indazole, an indole analog which has a nitrogen atom at the 2-position (pyrazole ring). The reaction product was isolated and identified as beta-indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H- and 13C-signals with several NMR techniques revealed that the beta-carbon of the product alanine moiety was bound to the 1-N-position of the indazole ring. This is the first example of the beta-replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3-position of indole analogs.     

复方大黄灌肠液对尿毒症患者的作用

目的 探讨复方大黄灌肠液对尿毒症患者肠道菌群、血清内毒素及炎症因子的影响.方法 选取2019年6月至2020年4月我院收治的90例尿毒症患者为研究对象,入选患者随机分为常规组与观察组,各45例.所有患者均采用常规治疗(血液透析,降压、降糖、调节饮食,改善贫血),观察组患者在此基础上给予复方大黄灌肠液进行灌肠.对比治疗前...     

Peroxidase catalyzed nitration of tryptophan derivatives. Mechanism, products and comparison with chemical nitrating agents.

The enzymatic nitration of tryptophan derivatives by oxidation of nitrite has been studied using lactoperoxidase and horseradish peroxidase, and compared with the chemical nitration produced by nitrogen dioxide and peroxynitrite. HPLC, mass spectra and NMR analysis of the mixture of products clearly show that nitration occurs at position 4-, 6-, 7-, and N1 of the indole ring, and nitrosation at position N1. Kinetic studies performed on peroxidase/NO2-/H2O2 systems showed substrate saturation behavior with all the tryptophan derivatives employed. The rate dependence on nitrite concentration was found to be linear with horseradish peroxidase while it exhibited saturation behavior with lactoperoxidase. The composition of the product mixture depends on the nitrating agent. While the production of 4-nitro, 6-nitro, 7-nitro and N1-nitro derivatives follows a similar trend, indicating that they are formed according to a similar mechanism, the ratio between the N1-nitroso derivative and other derivatives depends markedly on the nitrite concentration when tryptophan modification is performed by the peroxidase/H2O2/nitrite systems. Analysis of the data indicates that at low nitrite concentration the enzymatic reaction occurs through the classical peroxidase cycle. At high nitrite concentration the reaction proceeds through a different intermediate that we assume to be a protein bound peroxynitrite species.     

中枢神经系统感染患者脑脊液和血清中MMP-2、MMP-9、MCP-1表达的研究

目的探讨中枢神经系统感染患者脑脊液和血清中MMP-2(基质金属蛋白2)、MMP-9(基质金属蛋白9)、MCP-1(单核细胞趋化蛋白-1)表达的意义。方法选取2012年12月至2014年5月我院收治的中枢神经系统感染患者60例,其中结核性脑膜炎组(TBM组)、化脓性脑膜炎组(PM组)、真菌性脑膜炎组(CM组)和病毒性脑膜炎组(VM组)各15例;另外选取同期健康体检者15例作为对照组。治疗前、治疗后检测5组患者脑脊液常规及生化,采用ELISA法检测其脑脊液及血清中MMP-2、MMP-9、MCP-1的表达,并对患者进行格拉斯哥昏迷评分(GCS),分析其内在联系。结果治疗前、治疗后TBM组、PM组、CM组和VM组的脑脊液细胞数、蛋白水平均高于对照组,且TBM组、PM组的脑脊液细胞数、蛋白水平均高于CM组和VM组(P0.05);治疗前、治疗后TBM组、PM组、CM组和VM组的脑脊液糖水平、GCS评分低于对照组,治疗前TBM组、PM组、CM组、VM组的脑脊液氯化物水平低于对照组(P0.05);治疗后四组与对照组在脑脊液氯化物水平以及GCS评分等方面的差异无统计学意义(P0.05);治疗前、治疗后TBM组、PM组、CM组、VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平均显著高于对照组,且治疗前TBM组、PM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平均显著高于CM组、VM组以及对照组(P0.05);治疗后PM组、CM组、VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1水平的差异无统计学意义(P0.05);经Pearson进行相关性分析,BM组、PM组、CM组以及VM组的脑脊液和血清中MMP-2、MMP-9、MCP-1表达均与GCS评分负相关(相关系数r=-0.859~-0.574,P0.05)。结论中枢神经系统感染患者脑脊液和血清中MMP-2、MMP-9、MCP-1表达均与GCS评分相关,动态检测脑脊液和血清中MMP-2、MMP-9、MCP-1表达均对中枢神经系统感染的诊断和判断预后有积极意义。