Isodiospyrin as a novel human DNA topoisomerase I inhibitor

Isodiospyrin is a natural product from the plant Diospyros morrisiana, which consists of an asymmetrical 1,2-binaphthoquinone chromophore. Isodiospyrin exhibits cytotoxic activity to tumor cell lines but very little is known about its cellular target and mechanism of action. Unlike the prototypic human topoisomerase I (htopo I) poison camptothecin, isodiospyrin does not induce htopo I-DNA covalent complexes. However, isodiospyrin antagonizes camptothecin-induced, htopo I-mediated DNA cleavage. Binding analysis indicated that isodiospyrin binds htopo I but not DNA. These results suggest that isodiospyrin inhibits htopo I by direct binding to htopo I, which limits htopo I access to the DNA substrate. Furthermore, isodiospyrin exhibits strong inhibitory effect on the kinase activity of htopo I toward splicing factor 2/alternate splicing factor in the absence of DNA. Thus, these findings have important implications on naphthoquinone and its derivatives' cellular mode of actions, i.e. these novel DNA topoisomerase I inhibitors can prevent both DNA relaxation and kinase activities of htopo I.     

Naphthoquinones of Diospyros usambarensis; their Molluscicidal and Fungicidal Activities1

The molluscicidal and fungicidal properties of DIOSPYROS USAMBARENSIS root bark have been attributed to the naphthoquinone 7-methyljuglone. In addition, the dimeric naphthoquinones isodiospyrin and mamegakinone and 2 artefacts, 2-methoxy- and 3-methoxy-7-methyljuglone have been isolated.     

三磷酸腺苷结合转运蛋白G超家族成员2在肺癌A549和SPC-A-1细胞株中的表达及其意义

目的探讨三磷酸腺苷结合转运蛋白G超家族成员2（ABCG2）在肺癌细胞株A549和SPC—A-1中的表达及其意义。方法应用半定量逆转录-聚合酶链反应（RT—PCR）检测两种肺癌细胞株中该基因的表达，应用流式细胞仪检测该蛋白的表达。结果ABCG2基因与蛋白在肺腺癌细胞株A549和SPC—A-1中均有表达，且A549的表达高于SPC—A-1。结论ABCG2可能参与肺癌耐药，可能也与肺癌的发生、发展有密切相关。     

重组人血清白蛋白突变体干扰素2b融合蛋白的纯化

在成功构建人血清白蛋白突变体干扰素α2b融合蛋白(rmHSA-IFN-α2b)毕赤酵母工程菌株PicZαA/rmHSA-IFN-α2b/X33的基础上,建立了发酵分泌表达rmHSA-IFN-α2b的纯化工艺。对工程菌进行9 L罐高密度发酵后,离心收集发酵液上清,rmHSA-IFN-α2b的表达量为421 mg/L。对发酵液上清,依次进行3步纯化。第一步采用SP sepharose FF除去大部分色素和部分杂蛋白。第二步采用Blue sepharose FF根据亲和作用原理除去部分杂质。第三步采用Q sepharose FF除去溶剂和其他杂质以及类毒素。最终得到高纯度、有活性的rmHSA-IFN-α2b。通过HPLC检测和SDS-PAGE检测,最终产品的纯度都大于95%;通过活性检测,rmHSA-IFN-α2b的比活为1.5×106IU/mg。纯化总收率为25.3%。连续重复纯化6批,结果稳定。该工艺简单稳定,容易放大,适合规模化生产。简单高效纯化工艺的建立,为rmHSA-IFN-α2b后续研究奠定了坚实的基础。     

Plasma protein binding of the investigational anticancer agent 2-methoxyestradiol

2-Methoxyestradiol (2ME2) is an endogenously produced metabolite of estradiol currently being tested in phase I and II clinical trials as an anticancer agent. Here, we examined the role of protein binding as a possible determinant of the pharmacokinetic behavior of 2ME2. The distribution of 2ME2 in plasma was studied in vitro using plasma from healthy human volunteers and ex vivo using plasma from patients with cancer receiving the drug orally. The equilibrium dialysis method used to characterize plasma protein binding of 2ME2 utilized a tracer amount of [H]-2-methoxyestradiol on a 96-well microdialysis plate with a 5-kDa cutoff membrane and 250 mul of plasma. The time to equilibrium was approximately 24 h and the mean unbound fraction of 2ME2 (fu) over the observed concentration range in plasma of patients receiving 2ME2 orally was 0.019+/-0.0043. The mean fu was 0.027+/-0.0019 in plasma of healthy human volunteers. The binding was concentration independent, indicating a low-affinity, possibly nonspecific and nonsaturable process. The binding was also unaffected by the presence of 2-methoxyestrone, one of the major metabolites of 2ME2. 2ME2 was found to bind in decreasing order to plasma>albumin>alpha1-acid glycoprotein>sex-hormone-binding globulin. Plasma concentration-time profiles of total 2ME2 and unbound 2ME2 concentrations in a patient with cancer receiving 2ME2 as a single oral dose were parallel to each other. Thus, indicating that plasma protein binding is not an important consideration in pharmacokinetic monitoring of 2ME2.     

一种快速分离纯化金葡菌肠毒素C2的方法

Objective To develop a simple and convenient method to purify staphylococcal enterotoxin C₂ (SEC₂) from the supernatant of staphylococcus aureus culture. Methods SEC2was purified by ultrafiltration, anion ion-exchange chromatography, and cation ion-exchange chromatography sequentially. The molecular weight of SEC₂ was detected by the SDS-PAGE and flight time mass-spectrum . The purity of SEC₂ was detected by the SDS-PAGE and high performance liquid chromatography. The purified SEC2 was verified by amino acids sequence analysis. Results The purity of purified SEC₂ was greater than 98% (w)which was consistent with literature, including microheterogeneity of isoelectric point, pI 7.49 and 6.74, the molecular weigh 27.58 KD.The amino acids sequence of purified SEC2 was consistent with what was reported in literatures（ESQPDPTPDELHKSS）. The maximum absorption wavelength of purified SEC2 was 277.5 nm. Conclusions The method is rapid, simple and convenient with high SEC₂ purity, the recovery is more than 50 %(w).This method is suitable to large scale preparation of SEC₂.     

Preparation and properties of S-nitroso-L-cysteine ethyl ester, an intracellular nitrosating agent

In this report, a protocol for the preparation of the hydrochloride of S-nitroso-L-cysteine ethyl ester (SNCEE.HCl; 2) is presented. The synthesis of 2 has been targeted because S-nitroso-L-cysteine (SNC; 2b), which is extensively used for trans-S-nitrosation of thiol-containing proteins, has a limited ability of crossing cellular membranes. The nitrosothiol 2 was prepared via direct S-nitrosation of the hydrochloride of L-cysteine ethyl ester (CEE.HCl; 1a) with ethyl nitrite. 2 is relatively stable in crystal form and when neutralized to SNCEE (2a) in aqueous solutions treated with chelators of metal ions. Traces of metal ions, however, triggered the decomposition of 2a to nitric oxide and a S-centered radical, which were detected by ESR spectrometry. In contrast to 2b, 2a is a lipophilic compound that was taken up by human neutrophils. The latter process was paralleled by inhibition of the NADPH oxidase-dependent generation of superoxide anion radicals, presumably via reaction(s) of intracellular trans-S-nitrosation. Intracellular accumulation of S-nitrosothiols was observed with 2a but not with 2b. It is expected that the use of 2a will be advantageous when intracellular reactions of trans-S-nitrosation are to be studied.     

Metabolism of 2,3-dichloro-1-propene in the rat. Consideration of bioactivation mechanisms

At the present time, comprehensive metabolism studies of 2,3-dichloro-1-propene (2,3-DCP) have not yet been reported. We have investigated the biotransformation of 2,3-DCP using female Wistar rats in order to elucidate the bioactivation mechanisms. 175 mg/kg, 1,3-14C-2,3-DCP in corn oil was administered to a rat. The animal was killed 20 hr later. Approximately 56.7% of the radioactivity was excreted in the urine, 1.6% in the feces, 5.3% was exhaled as unchanged 2,3-DCP, and 0.3% as CO2. 31.3% remained in the organs and the carcass. Three metabolic pathways were established. 1) Conjugation with GSH leading to S-(2-chloro-2-propenyl)mercapturic acid. 2) The P450 induced epoxidation with subsequent rearrangement to highly mutagenic 1,3-dichloroacetone. 1,3-Dichloroacetone was further converted to the dimercapturic acid, 1,3-(2-propanone)-bis-S-(N-acetylcysteine). 3) The hydrolysis to 2-chloroallyl alcohol followed by alcohol dehydrogenase catalyzed formation of highly mutagenic 2-chloroacrolein. The 2-chloroallyl alcohol is excreted directly in the urine and as the glucuronide. 2-Chloroacrolein is further oxidized to 2-chloroacrylic acid which is also excreted in the urine.     

Comparison of cytochrome P4502E1 (CYP2E1) activity and hepatic and lymphocyte mRNA expression in patients with chronic hepatitis C

The induction of cytochrome P4502E1 (CYP2E1) is believed to play a role in the development of fibrosis in hepatitis C patients. However, information about CYP2E1 activity in chronic hepatitis C patients is fragmentary and the relationship between CYP2E1 activity and mRNA expression is unknown in this disease. The purpose of this study was (a) to characterise CYP2E1 activity in those patients and (b) to analyse its relationship with CYP2E1 mRNA expression in the liver and in peripheral blood lymphocytes (PBLs), previously proposed as a surrogate to assess changes in CYP2E1 activity. Fourteen chronic hepatitis C patients were submitted to a routine transcutaneous liver biopsy. CYP2E1 activity was assessed by using chlorzoxazone (CZX) pharmacokinetic parameters and hepatic and PBLs CYP2E1 mRNA expression was measured by real-time RT-PCR. The mean oral clearance of CZX (CLT: 21.5+/-10.1L/h) was within the normal range and the chlorzoxazone metabolic ratio (CMR) at t = 2 h was closely related to other CZX pharmacokinetic parameters. None of the pharmacokinetic parameters did significantly correlate with CYP2E1 mRNA, neither in the liver nor in PBLs. Furthermore, there was no significant relationship between CYP2E1 mRNA levels in paired liver and PBL samples. Our data indicate that early stages of chronic hepatitis C are not associated with CYP2E1 induction. In this disease, the determination of the CMR at t = 2 h represents a reliable index to assess CYP2E1 activity. The measurement of CYP2E1 expression, at the mRNA level, in PBLs or in liver is not useful for that purpose.     

温度对采用HPLC法精制白细胞介素-2的影响

目的分析在不同柱温状态下HPLC法对制备rhIL-2收率的影响。方法将rhIL-2复性液以50ml.min-1的速度进样,以不同的乙腈浓度进行洗脱,以280nm紫外分光光度法检测分析,并分析在不同的柱温状态下HPLC法对制备rhIL-2的收率影响。结果与结论采用HPLC法制备重组人白细胞介素-2时,有效的控制温度有利于rhIL-2收率,当柱温为24℃—28℃时蛋白收率较高,纯度符合要求。     

A 3D structural model of memapsin 2 protease generated from theoretical study.

AIM: To build a 3D structural model of memapsin 2 (M2) protease for theoretical study and drug design. METHODS: Structural alignment was performed based on multiple and pairwise sequence alignment of three templates. After the initial model was generated, energy minimization was completed by applying molecular mechanics method. Molecular dynamics (MD) technique was used to do further structural optimization. RESULTS: The 3D structural model of memapsin 2 was constructed. The model is reasonable according to several validation criteria. The active-site motifs of M2 are structurally supported by a beta-sheet rich domain and linked together with this domain through alpha helices. Tyr132 contained in beta-hairpin is a general characteristic of aspartic protease. The Calpha atom superimposing result is a direct verification that M2 is structurally unique but still belongs to the aspartic protease superfamily. CONCLUSION: The 3D-structure model from our study is informative to guide future molecular biology study about M2 and drug design based on database searching.     

Initial study on apoptosis in HepG-2 Human heptocarcinoma cell line by CSS

Objective To discuss on mechanism of the killing and apoptosis inducing effect induced by total alkaloid in the CSS(Capparis spinosa L.saponin,CSS)on human hepatocarcinoma cell Line HepG-2.Methods The killing effect of the CSS on human hepatocarcinoma cell Line HepG-2 was observed by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.This test was signed to observe the changes of the cell cycle of HepG-2 cells affected by the CSS by PI single-staining,and to observe if there were typical apoptosis peaks.The apoptosis inducing effect and changing of mitochondria membrane potential of the CSS on the HepG-2 cells were studied by flow cytometry.The effect of intracellular Ca2+ level of CSS on the HepG-2 cells was measured by laser confocal microscope.Results CSS has growth inhibiting on the HepG-2 and seems to be enhanced with the increasing concentration of CSS,and its IC50 value was 46.16 μg·mL-1.The HepG-2 cells are characteristic apoptosis morphologic changed,and the apoptosis percentage is increased to 66.652% in the 50 μg·mL-1 dosage group.The cells cycle has been changed obviously that the progresses of cells cycle of G1 period and G2 period in high dosage group have been blocked,and the cellular proportion in G2 period is decreased by the function of CSS for 24 h.The mitochondria membrane potential of HepG-2 cells induced by CSS is decreased in various degrees.In addition,the intracellular Ca2+ level is increased by the function of CSS in the middle and high dose groups.Conclusions The CSS has obviously killing and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.     

The fate of 2-chlorobenzylidene malononitrile (CS) in rats

1. The fate of 3H-ring labelled, 14C-cyanide labelled and (14C = C) side chain labelled 2-chlorobenzylidene malononitrile (CS), 2-chlorobenzyl alcohol and 2-chlorobenzyl malononitrile (dihydro CS) in rats and isolated rat liver cells has been examined. 2. CS was administered both i.v. and i.g. to rats at doses from 0.08 to 159 mumol/kg and in most cases the greatest proportion of the dose was eliminated in the urine (44-100%). The principal urinary metabolites were 2-chlorohippuric acid, 1-O-(2-chlorobenzyl) glucuronic acid, 2-chlorobenzyl cysteine and 2-chlorobenzoic acid. Lesser amounts of 2-chlorophenyl acetyl glycine, 2-chlorobenzyl alcohol and 2-chlorophenyl 2-cyano propionate were identified. 3. The major urinary metabolite from 2-chlorobenzyl alcohol was 2-chlorohippuric acid (43%), 2-chlorobenzyl cysteine, 2-chlorobenzoic acid and 2-chlorobenzyl glucuronic acid were identified. 4. The products of dihydro-CS metabolism were 2-chlorophenyl acetyl glycine, 2-chlorophenyl 2-cyanopropionate and 2-chlorophenyl proprionamide. 5. Urinary thiocyanate levels increased with the dose of CS up to 159 mumol/kg. At 212 mumol/kg there was a large increase in the amount of thiocyanate produced (molar conversion: 21.5-29.9%). Similarly malononitrile, the hydrolysis product of CS, gave a dose related increase in urinary thiocyanate levels. However at a higher dose (212 mumol/kg) the molar conversion was greater than 60%. 6. The metabolism of CS by isolated rat liver cells confirmed the results in vivo but demonstrated a marked limitation of this preparation to form conjugates. 7. It is concluded that CS in vivo is hydrolysed mainly to 2-chlorobenzaldehyde which is then either oxidized to 2-chlorobenzoic acid for subsequent glycine conjugation, or reduced to 2-chlorobenzyl alcohol for ultimate excretion as 2-chlorobenzyl acetyl cysteine or 1-O-(2-chlorobenzyl) glucuronic acid. Malononitrile is converted to thiocyanate via the formation of cyanide.     

The metabolism of 17 alpha-ethinyloestradiol by human liver microsomes: formation of catechol and chemically reactive metabolites.

The metabolism of 17 alpha-ethinyloestradiol (EE2) to catechol and reactive metabolites by human liver microsomes was investigated. 2-Hydroxyethinyloestradiol (2-OHEE2) was either the sole or principal metabolite. Small amounts of 6-hydroxyethinyloestradiol and 16-hydroxyethinyloestradiol were produced by some of the livers. EE2 (10 microM) underwent substantial (5-20% of incubated drug), though highly variable, NADPH-dependent metabolism to material irreversibly bound to microsomal protein. 2-OHEE2 appeared to be the pro-reactive metabolite. The maximum EE2 2-hydroxylase activity was 0.67 nmol min-1 mg-1 microsomal protein, with a Km value of 8.6 microM. Oestradiol, which is mainly hydroxylated to 2-hydroxyoestradiol, was the most potent inhibitor of hydroxylase activity and exhibited competitive inhibition. Progesterone, which undergoes 2-hydroxylation to a minor extent was also a competitive inhibitor, whereas cholesterol and cortisol did not have any appreciable inhibitory effect. Primaquine was the most potent non-steroidal inhibitor but was non-competitive. Other non-steroidal compounds investigated, e.g. antipyrine, did not show any significant effect on EE2 2-hydroxylation. The results of this study suggest that EE2 2-hydroxylation is metabolised by a form(s) of cytochrome P-450 which has affinity for endogenous steroids.     

Hydrogen peroxide reduces beta-adrenoceptor function in the rat small intestine.

Incubation of isolated rat intestinal segments with hydrogen peroxide (H2O2) led to a decreased beta-adrenoceptor response. The maximal relaxation induced by isoprenaline was lowered while the EC50 remained unaffected. The effect of H2O2 in the small intestine increased slightly from duodenum to ileum. In the ileum, 10(-4) M H2O2 led to a 10% decrease of the maximal relaxation due to isoprenaline and 1 mM decreased the maximal response to about 50%. We further investigated the level at which the isoprenaline response was impaired. The relaxation caused by the stable cAMP analog, dibutyryl-cAMP, or by the adenylate cyclase activator, forskolin, was not affected or affected less than by isoprenaline. When the response to isoprenaline was expressed relative to the maximal response to dibutyryl-cAMP or forskolin, pretreatment with H2O2 led to a decreased isoprenaline response relative to the response to dibutyryl-cAMP or forskolin. This might indicate that exposure to H2O2 leads to a disturbance in receptor-mediated cAMP production. The adenylate cyclase unit is probably not affected since the response to forskolin is relatively resistant to H2O2. Our conclusion is that pretreatment of isolated intestinal segments with H2O2 leads to disturbed beta-adrenoceptor coupling, probably due to altered membrane integrity.     

Identification of a new urinary metabolite of carbon disulfide using an improved method for the determination of 2-thioxothiazolidine-4-carboxylic acid

A new method is reported for the analysis of 2-thioxothiazolidine-4-carboxylic acid (TTCA) in urine that is amenable to automation and provides greatly simplified chromatograms. The method comprises the addition of tetrahydro-2-thioxo-2H-1,3-thiazine-4-carboxylic acid, which is chemically similar to TTCA, as internal standard, purification on an Oasis HLB solid-phase extraction column, and analysis by HPLC with UV detection. The limit of detection for TTCA was 40 pmol/mL of urine, recovery was 79.3 +/- 1.0%, and detection was linear over at least 3 orders of magnitude. In addition, during the analysis of urine samples from workers exposed to CS(2), a novel urinary metabolite of CS(2) was recognized. The new metabolite demonstrated a dose response, was present at approximately 30% the level of TTCA, and was charaterized to be 2-thioxothiazolidin-4-ylcarbonylglycine (TTCG). Administration of TTCG to rats resulted in excretion of TTCA suggesting that TTCG is a likely precursor of TTCA. Although urinary excretion of both TTCA and TTCG resulted from administration of captan, only TTCA was detected following administration of methyl isothiocyanate. The greater selectivity of TTCG suggests that co-analysis of TTCA and TTCG in urine may aid in differentiating exposures to CS(2), captan and isothiocyanates.     

Metabolites of 2-chlorosyringaldehyde in fish bile: indicator of exposure to bleached hardwood effluent

1. 2-Chlorosyringaldehyde (2-CSA) is the major chlorinated phenol produced by the 100% chlorine dioxide bleaching of eucalypt pulp and is found in other bleached hardwood effluents. Almost nothing is known of the environmental or metabolic fates of this chemical.

2. Sand flathead (Platycephalus bassensis) was given 2-CSA by intraperitoneal injection at 0.15,1.5, 15 and 75?mg/kg doses and, 4 days later, bile was collected and solvent extracted before and after enzymatic cleavage of conjugates. The acetate derivatives of bile extracts were analysed by gas chromatography/mass spectrometry.

3. The major metabolite 4 days after administration was the glucuronide or sulphate conjugate of 2-chloro-4-hydroxy-3,5-dimethoxy-benzylalcohol (2-CB-alcohol). The identity of 2-CB-alcohol was confirmed by chemical synthesis.

4. The quantity of 2-CB-alcohol in the bile was linearly related to dose of 2-CSA and was detected at all dose levels. Minor metabolites identified were conjugated 2-CSA. unchanged 2-CSA and 2-chloro-4-hydroxy-3,5-dimethoxy-benzoic acid.

5. The amount of 2-CB-alcohol in bile has the potential to be a sensitive and specific indicator of fish exposure or bleached hardwood effluent.     

顶空气相色谱法测定氨苄西林钠舒巴坦钠中残留溶媒-乙醇、异丙醇

目的：建立一种气相色谱法同时测定氨苄西林钠舒巴坦钠中有机溶剂乙醇、异丙醇残留量的方法。方法：采用外标法项空自动进样．在非极性弹性毛细管柱上进行分离,效果良好。结果：平均回收率乙醇96．9％～104％;异丙醇96．3％～101．2％。变异系数乙醇2．6％。异丙醇2．7％。结论：方法重现性好,定量准确,便于操作。     

Metabolic activation of Trp-P-2, a tryptophan-pyrolysis mutagen, by isolated rat hepatocytes

Metabolic activation of a tryptophan-pyrolysis product, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), by isolated rat hepatocytes was studied. The substrate (Trp-P-2) disappearance by hepatocytes from untreated rats was slow, but enhanced by 3-methylcholanthrene (MC) pretreatment of rats. The covalent binding of Trp-P-2 to cellular macromolecules was detected in hepatocytes from untreated rats. The amount of covalent binding of Trp-P-2 to protein and RNA was greater than that to DNA. The covalent binding to Trp-P-2 to DNA, RNA and protein in hepatocytes from untreated rats was about 5-10 times less than that in hepatocytes from MC-pretreated rats. 7,8-Benzoflavone strongly inhibited the substrate disappearance and the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. These results indicate that Trp-P-2 is metabolically activated by the P-448 type of cytochrome P-450 which is induced by MC. Diethylmaleate enhanced by about 50% the binding of Trp-P-2 to DNA in hepatocytes from MC-pretreated rats. On the other hand, cysteine inhibited the binding of Trp-P-2 to DNA with a concomitant reduction in the accumulation of the active metabolite, N-hydroxy-Trp-P-2 (N-OH-Trp-P-2). Sulfhydryl compounds seemed to play important roles in the detoxification of Trp-P-2.     

Mechanism of the reaction of ebselen with endogenous thiols: dihydrolipoate is a better cofactor than glutathione in the peroxidase activity of ebselen

The therapeutic effect of ebselen has been linked to its peroxidase activity. In the present study, the peroxidase activity of ebselen toward H2O2 with the endogenous thiols GSH and dihydrolipoate [L(SH)2] as cofactors was determined. When GSH was used, peroxide removal was described by a ter uni ping pong mechanism with Dalziel coefficients for GSH and H2O2 of 0.165 +/- 0.011 and 0.081 +/- 0.005 mM min, respectively. When L(SH)2 was used, peroxidase activity was independent of the concentration of L(SH)2 in the concentration range studied (5 microM to 2 mM) and peroxide removal was only dependent on the concentration of H2O2 and ebselen, with the second-order rate constant being 12.3 +/- 0.8 mM-1 min-1. To elucidate the difference between GSH and L(SH)2, the molecular mechanism of the peroxidase activity of ebselen was investigated, using UV spectrophotometry, high pressure liquid chromatography, 77Se NMR, and mass spectrometry. GSH was found to react quickly with ebselen to give a selenenyl sulfide, an adduct of GSH to ebselen. Subsequently, the GSH-selenenyl sulfide is converted into the diselenide of ebselen. Finally the diselenide reacts with a peroxide and ebselen is regenerated. The formation by GSH of the diselenide from the GSH-selenenyl sulfide of ebselen is slow and linearly dependent on the concentration of free thiol; however, no net consumption of GSH was observed. Furthermore, it is likely that a selenol is an intermediate in diselenide formation. After reaction between ebselen and L(SH)2 the diselenide of ebselen was immediately detected. The fast formation of the diselenide with L(SH)2 versus the slow formation of the diselenide with GSH accounts for our observation that L(SH)2 is a better cofactor than GSH in the peroxidase activity of ebselen. Our results suggest that the interaction between ebselen and L(SH)2 might be of major importance in the mechanism by which ebselen exerts its therapeutic effect.