Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.

Transport of UDP-rhamnose by URGT2, URGT4, and URGT6 modulates rhamnogalacturonan-I length.

Rhamnogalacturonan-I biosynthesis occurs in the lumen of the Golgi apparatus, a compartment where UDP-Rhamnose and UDP-Galacturonic Acid are the main substrates for synthesis of the backbone polymer of pectin. Recent studies showed that UDP-Rha is transported from the cytosol into the Golgi apparatus by a family of six UDP-rhamnose/UDP-galactose transporters (URGT1-6). In this study, analysis of adherent and soluble mucilage (SM) of Arabidopsis thaliana seeds revealed distinct roles of URGT2, URGT4, and URGT6 in mucilage biosynthesis. Characterization of SM polymer size showed shorter chains in the urgt2 urgt4 and urgt2 urgt4 urgt6 mutants, suggesting that URGT2 and URGT4 are mainly involved in Rhamnogalacturonan-I (RG-I) elongation. Meanwhile, mutants in urgt6 exhibited changes only in adherent mucilage (AM). Surprisingly, the estimated number of RG-I polymer chains present in urgt2 urgt4 and urgt2 urgt4 urgt6 mutants was higher than in wild-type. Interestingly, the increased number of shorter RG-I chains was accompanied by an increased amount of xylan. In the urgt mutants, expression analysis of other genes involved in mucilage biosynthesis showed some compensation. Studies of mutants of transcription factors regulating mucilage formation indicated that URGT2, URGT4, and URGT6 are likely part of a gene network controlled by these regulators and involved in RG-I synthesis. These results suggest that URGT2, URGT4, and URGT6 play different roles in the biosynthesis of mucilage, and the lack of all three affects the production of shorter RG-I polymers and longer xylan domains.

Gene Mining and Sequence Analysis of Purine Nucleosidase Based on RNA-Seq.

Caterpillar fungus (Ophiocordyceps sinensis) is one of the most valued fungal Traditional Chinese medicine (TCM), and it contains plenty of active ingredients such as adenosine. Adenosine is considered as a biologically effective ingredient that has a variety of anti-tumor and immunomodulatory activities. In order to further elucidate the mechanism of purine nucleosidase (PN) in adenosine biosynthesis, a gene encoding PN was successfully mined and further analyzed based on the RNA-Seq database of caterpillar fungus. The full-length cDNA of PN was 855 bp, which encoded 284 amino acids. BLAST analysis showed the highest homology of 85.06% with nucleoside hydrolase in NCBI. ProtProm analysis showed that the relative molecular weight was 30.69 kDa and the isoelectric point was 11.55. The secondary structure of PN was predicted by Predict Protein; the results showed that alpha helix structure accounted for 28.17%, strand structure accounted for 11.97%, and loop structure accounted for 59.86%. Moreover, PN gene was further cloned from transcriptome and detected by agarose gel electrophoresis for verification. This study provides more sufficient scientific basis and new ideas for the genetic regulation of adenosine biosynthesis in fungal TCM.

Purification, characterization, and functional properties of a novel glycoprotein from tartary buckwheat (Fagopyrum tartaricum) seed.

A pure glycoprotein (BGP4-I) was obtained from tartary buckwheat seeds by aqueous extraction followed by DEAE-Sepharose Fast Flow ion exchange chromatography and Sephadex G-100 gel filtration chromatography. The average molecular weight of BGP4-I, as determined by high performance gel permeation chromatography, was 123.43 kDa. The structure of BGP4-I was characterized based on Fourier transform infrared spectroscopy, circular dichroism spectroscopy, and nuclear magnetic resonance spectroscopy, etc. Based on the nano-liquid chromatography-coupled electrospray ionization mass spectrometry analysis of the amino acid sequence of BGP4-I, belongs unequivocally to the glycosyl hydrolase family 1 in the Carbohydrate Active Enzymes database by alignment studies. The specific activity of BGP4-I was 18.44 µmol/min/mg on the substrate p-nitrophenyl-ß-d-glucopyranoside. Furthermore, BGP4-I is unique in its specificity for some substrates. These results suggest that the BGP4-I from tartary buckwheat seeds is a novel specific ß-glucosidase setting the foundation for potential applications in the food industry.

Ginsenoside Rd Attenuates DNA Damage by Increasing Expression of DNA Glycosylase Endonuclease VIII-like Proteins after Focal Cerebral Ischemia.

BACKGROUND: Ginsenoside Rd (GSRd), one of the main active ingredients in traditional Chinese herbal Panax ginseng, has been found to have therapeutic effects on ischemic stroke. However, the molecular mechanisms of GSRd's neuroprotective function remain unclear. Ischemic stroke-induced oxidative stress results in DNA damage, which triggers cell death and contributes to poor prognosis. Oxidative DNA damage is primarily processed by the base excision repair (BER) pathway. Three of the five major DNA glycosylases that initiate the BER pathway in the event of DNA damage from oxidation are the endonuclease VIII-like (NEIL) proteins. This study aimed to investigate the effect of GSRd on the expression of DNA glycosylases NEILs in a rat model of focal cerebral ischemia. METHODS: NEIL expression patterns were evaluated by quantitative real-time polymerase chain reaction in both normal and middle cerebral artery occlusion (MCAO) rat models. Survival rate and Zea-Longa neurological scores were used to assess the effect of GSRd administration on MCAO rats. Mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damages were evaluated by the way of real-time analysis of mutation frequency. NEIL expressions were measured in both messenger RNA (mRNA) and protein levels by quantitative polymerase chain reaction and Western blotting analysis. Apoptosis level was quantitated by the expression of cleaved caspase-3 and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling assay. RESULTS: We found that GSRd administration reduced mtDNA and nDNA damages, which contributed to an improvement in survival rate and neurological function; significantly up-regulated NEIL1 and NEIL3 expressions in both mRNA and protein levels of MCAO rats; and reduced cell apoptosis and the expression of cleaved caspase-3 in rats at 7 days after MCAO. CONCLUSIONS: Our results indicated that the neuroprotective function of GSRd for acute ischemic stroke might be partially explained by the up-regulation of NEIL1 and NEIL3 expressions.

A novel nucleoside from the edible mushroom, Tricholoma japonicum.

A novel nucleoside, 9-ß-D-ribopyranosylpurine (2), along with three known nucleosides, adenosine (1), uridine (3) and nebularine (4), were isolated from the edible mushroom, Tricholoma japonicum. The structure of 2 was determined as 9-ß-D-ribopyranosylpurine by comparing the reported spectral data of 2 with that of a synthetic compound. Isolation of the glycoside, which contains the sugar ribopyranose, from natural resources is very unusual. There are reports on the synthesis of 9-ß-D-ribopyranosylpurine (2), but this is the first report on the isolation from natural resources. The antiproliferative activity of compounds 1-4 was evaluated using human umbilical vein endothelial cells. Compound 4 showed the highest inhibitory activity.

Transcriptomic analysis of Camellia ptilophylla and identification of genes associated with flavonoid and caffeine biosynthesis.

Camellia ptilophylla, or cocoa tea, is naturally decaffeinated and its predominant catechins and purine alkaloids are trans-catechins and theobromine Regular tea [Camellia sinensis (L.) O. Ktze.] is evolutionarily close to cocoa tea and produces cis-catechins and caffeine. Here, the transcriptome of C. ptilophylla was sequenced using the 101-bp paired-end technique. The quality of the raw data was assessed to yield 70,227,953 cleaned reads totaling 7.09 Gbp, which were assembled de novo into 56,695 unique transcripts and then clustered into 44,749 unigenes. In catechin biosynthesis, leucoanthocyanidin reductase (LAR) catalyzes the transition of leucoanthocyanidin to trans-catechins, while anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR) catalyze cis-catechin production. Our data demonstrate that two LAR genes (CpLAR1 and CpLAR2) by C. ptilophylla may be advantageous due to the combined effects of this quantitative trait, permitting increased leucoanthocyanidin consumption for the synthesis of trans-catechins. In contrast, the only ANS gene observed in C. sinensis (CsANS) shared high identity (99.2%) to one homolog from C. ptilophylla (CpANS1), but lower identity (~80%) to another (CpANS2). We hypothesized that the diverged CpANS2 might have lost its ability to synthesize cis-catechins. C. ptilophylla and C. sinensis each contain two copies of ANR, which share high identity and may share the same function. Transcriptomic sequencing captured two N-methyl nucleosidase genes named NMT1 and NMT2. NMT2 was highly identical to three orthologous genes TCS2, PCS2, and ICS2, which did not undergo methylation in vitro; in contrast, NMT1 was less identical to TCS, PCS and ICS, indicating that NMT1 may undergo neofunctionalization.

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte.

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.

A cell wall-bound adenosine nucleosidase is involved in the salvage of extracellular ATP in Solanum tuberosum.

Extracellular ATP (eATP) has recently been demonstrated to play a crucial role in plant development and growth. To investigate the fate of eATP within the apoplast, we used intact potato (Solanum tuberosum) tuber slices as an experimental system enabling access to the apoplast without interference of cytosolic contamination. (i) Incubation of intact tuber slices with ATP led to the formation of ADP, AMP, adenosine, adenine and ribose, indicating operation of apyrase, 5'-nucleotidase and nucleosidase. (ii) Measurement of apyrase, 5'-nucleotidase and nucleosidase activities in fractionated tuber tissue confirmed the apoplastic localization for apyrase and phosphatase in potato and led to the identification of a novel cell wall-bound adenosine nucleosidase activity. (iii) When intact tuber slices were incubated with saturating concentrations of adenosine, the conversion of adenosine into adenine was much higher than adenosine import into the cell, suggesting a potential bypass of adenosine import. Consistent with this, import of radiolabeled adenine into tuber slices was inhibited when ATP, ADP or AMP were added to the slices. (iv) In wild-type plants, apyrase and adenosine nucleosidase activities were found to be co-regulated, indicating functional linkage of these enzymes in a shared pathway. (v) Moreover, adenosine nucleosidase activity was reduced in transgenic lines with strongly reduced apoplastic apyrase activity. When taken together, these results suggest that a complete ATP salvage pathway is present in the apoplast of plant cells.

Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+.

Although NAD(+) biosynthesis is required for Sir2 functions and replicative lifespan in yeast, alterations in NAD(+) precursors have been reported to accelerate aging but not to extend lifespan. In eukaryotes, nicotinamide riboside is a newly discovered NAD(+) precursor that is converted to nicotinamide mononucleotide by specific nicotinamide riboside kinases, Nrk1 and Nrk2. In this study, we discovered that exogenous nicotinamide riboside promotes Sir2-dependent repression of recombination, improves gene silencing, and extends lifespan without calorie restriction. The mechanism of action of nicotinamide riboside is totally dependent on increased net NAD(+) synthesis through two pathways, the Nrk1 pathway and the Urh1/Pnp1/Meu1 pathway, which is Nrk1 independent. Additionally, the two nicotinamide riboside salvage pathways contribute to NAD(+) metabolism in the absence of nicotinamide-riboside supplementation. Thus, like calorie restriction in the mouse, nicotinamide riboside elevates NAD(+) and increases Sir2 function.

Molecular characterization of the recombinant A-chain of a type II ribosome-inactivating protein (RIP) from Viscum album coloratum and structural basis on its ribosome-inactivating activity and the sugar-binding properties of the B-chain.

Mistletoe (Viscum album) lectins, which are classified as a type II ribosome-inactivating protein (RIP) due to their unique biological function and the potential medical and therapeutic application in cancer cells, receive a rising attention. The heterodimeric glycoproteins contain the Achain with catalytic activity and the B-chain with sugar binding properties. In recent years, studies involving the lectins from the white berry European mistletoe (Viscum album) and the yellow berry Korean mistletoe (Viscum album coloratum) have been described. However, the detailed mechanism in exerting unique cytotoxic effect on cancer cells still remains unclear. Here, we aim to understand and define the molecular basis and biological effects of the type II RIPs, through the studies of the recombinant Korean mistletoe lectin. To this end, we expressed, purified the recombinant Korean mistletoe lectin (rKML), and investigated its molecular characteristics in vitro, its cytotoxicity and ability to induce apoptotic cell death in cancer cells. To gain structural basis for its catalytic activity and sugar binding properties, we performed homology modeling studies based on the high degree of sequence identity and conserved secondary structure prediction between Korean and European, Himalayan mistletoe lectins, and Ricin.

Calcium-stimulated guanosine--inosine nucleosidase from yellow lupin (Lupinus luteus).

Guanosine-inosine-preferring nucleoside N-ribohydrolase has been purified to homogeneity from yellow lupin (Lupinus luteus) seeds by ammonium sulfate fractionation, ion-exchange chromatography and gel filtration. The enzyme functions as a monomeric, 80kDa polypeptide, most effectively between pH 4.7 and 5.5. Of various mono- and divalent cations tested, Ca(2+) appeared to stimulate enzyme activity. The nucleosidase was activated 6-fold by 2mM exogenous CaCl(2) or Ca(NO(3))(2), with K(a)=0.5mM (estimated for CaCl(2)). The K(m) values estimated for guanosine and inosine were 2.7+/-0.3 microM. Guanosine was hydrolyzed 12% faster than inosine while adenosine and xanthosine were poor substrates. 2'-Deoxyguanosine, 2'-deoxyinosine, 2'-methylguanosine, pyrimidine nucleosides and 5'-GMP were not hydrolyzed. However, the enzyme efficiently liberated the corresponding bases from synthetic nucleosides, such as 1-methylguanosine, 7-methylguanosine, 1-N(2)-ethenoguanosine and 1-N(2)-isopropenoguanosine, but hydrolyzed poorly the ribosides of 6-methylaminopurine and 2,6-diaminopurine. MnCl(2) or ZnCl(2) inhibited the hydrolysis of guanosine with I(50) approximately 60 microM. Whereas 2'-deoxyguanosine, 2'-methylguanosine, adenosine, as well as guanine were competitive inhibitors of this reaction (K(i) values were 1.5, 3.6, 21 and 9.7 microM, respectively), hypoxanthine was a weaker inhibitor (K(i)=64 microM). Adenine, ribose, 2-deoxyribose, 5'-GMP and pyrimidine nucleosides did not inhibit the enzyme. The guanosine-inosine hydrolase activity occurred in all parts of lupin seedlings and in cotyledons it increased up to 5-fold during seed germination, reaching maximum in the third/fourth day. The lupin nucleosidase has been compared with other nucleosidases.

Enzymatic activity of toxic and non-toxic type 2 ribosome-inactivating proteins.

Ribosome-inactivating proteins (RIPs) display adenine polynucleotide glycosylase activity on different nucleic acid substrates, which at the ribosomal level is responsible for the arrest of protein synthesis. Some type 2 RIPs, namely ricin and related proteins, are extremely toxic to mammalian cells and animals whilst other type 2 RIPs (non-toxic type 2 RIPs) display three to four logs less toxicity. We studied whether a correlation exists between toxicity on cells and enzymatic activity on nucleic acids. All type 2 RIPs differ in their depurinating activity on the different substrates with differences of up to one to two logs. The toxicity of type 2 RIPs is independent of their enzymatic activity on nucleic acids or on ribosomes.

One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.

The Arabidopsis FWA gene was initially identified from late-flowering epigenetic mutants that show ectopic FWA expression associated with heritable hypomethylation of repeats around transcription starting sites. Here, we show that wild-type FWA displays imprinted (maternal origin-specific) expression in endosperm. The FWA imprint depends on the maintenance DNA methyltransferase MET1, as is the case in mammals. Unlike mammals, however, the FWA imprint is not established by allele-specific de novo methylation. It is established by maternal gametophyte-specific gene activation, which depends on a DNA glycosylase gene, DEMETER. Because endosperm does not contribute to the next generation, the activated FWA gene need not be silenced again. Double fertilization enables plants to use such "one-way" control of imprinting and DNA methylation in endosperm.

Antitumor effects of curcin from seeds of Jatropha curcas.

AIM: To study the antitumor effects of curcin from Jatropha curcas. METHODS: Antitumor activity of curcin was tested by MTT assay. The N-glycosidase activity of curcin was determined by characterization of R-fragment in gel. A cell-free system, rabbit reticulocyte lysate, was introduced to quantify the inhibitory activity of curcin on protein biosynthesis. RESULTS: The curcin had a powerful inhibitory action upon protein synthesis in reticulocyte lysate with an IC50 (95 % confidence limits) value of 0.19 (0.11-0.27) nmol/L. The IC50 (95 % confidence limits) of curcin on SGC-7901, Sp2/0, and human hepatoma was 0.23 (0.15-0.32) mg/L, 0.66 (0.35-0.97) mg/L, 3.16 (2.74-3.58) mg/L, respectively. Curcin was found to have no toxic to Hela cells and normal cells (MRC). After the rRNA of ribosome was treated with curcin and aniline at acidic condition, a cleaved R-fragment of approximately 450 nt appeared, but this fragment did not occur after treatment with curcin only. A comparison of the amino acid sequences of curcin, ricin A-chain and trichosanthin revealed that there were relatively high similarities among them. The percentages of homology between curcin and ricin A chain, between curcin and trichosanthin were found to be 54 % and 57 % respectively. Especially, the conserved residues forming the active sites of the A chain of ricin and trichosanthin occurred in curcin. CONCLUSION: Curcin has an obvious antitumor effect and its mechanisms are related to the N-glycosidase activity.

One-electron oxidation of plasmid DNA by selenium(V) species.

PURPOSE: To employ the gamma-radiation-generated selenium(V) one-electron-oxidizing agent SeO3*- for the preparation of guanyl radicals in plasmid DNA, and to compare the behaviour of this reagent with that of other similarly reactive oxidant species. MATERIALS AND METHODS: Plasmid DNA in aerobic aqueous solution was irradiated with 137Cs gamma-rays (662 keV). The solutions also contained up to 4x10(-2) mol x dm(-3) sodium selenate (Na2SeO4) and/or up to 10(-1) mol x dm(-3) sodium biselenite (NaHSeO3), as well as auxiliary scavengers such as DMSO or glycerol. In some cases, reducing agents such as ferrocyanide were also present. After irradiation, the plasmid was incubated with the Escherichia coli base excision-repair endonuclease formamidopyrimidine-DNA N-glycosylase (FPG). These treatments produced strand breaks in the plasmid. The yields of these strand breaks were quantified by agarose gel electrophoresis. RESULTS: In general, gamma-irradiation produced single-strand breaks (SSB) in plasmid DNA. Subsequent incubation with the endonuclease FPG increased the SSB yield by a factor of 2-100-fold. The smallest effects of FPG were observed when only DMSO or glycerol were present during irradiation. FPG incubation produced significantly larger increases in the SSB yield after gamma-irradiation in the additional presence of selenate and/or biselenite. The largest effect of FPG was observed after gamma-irradiation in the presence of 10(-2) mol x dm(-3) sodium selenate and 10(-1) mol x dm(-3) glycerol. This was indicative of extensive oxidative damage to the plasmid under these conditions and provided evidence for guanine oxidation mediated by SeO3*-. The large effect of FPG was strongly attenuated by the addition of reducing agents such as ferrocyanide. The observations suggest that these reducing agents exert their effects through the reduction of an intermediate guanyl radical. CONCLUSION: By comparing the yields of breaks produced after gamma-irradiation under a range of conditions, it is possible to formulate a reaction scheme that describes the chemical reactions responsible for the formation of strand breaks and FPG-sensitive sites. By applying this scheme to the data, we can quantify rate constants for the reduction of DNA guanyl radicals by reducing agents. This reaction is of particular interest to radiation biology because it is the equivalent of the repair of DNA damage by the direct effect of ionizing radiation.

Functional interaction of MutY homolog with proliferating cell nuclear antigen in fission yeast, Schizosaccharomyces pombe.

The MutY homolog (MYH) is responsible for removing adenines misincorporated on a template DNA strand containing G or 7,8-dihydro-8-oxoguanine (8-oxoG) and thus preventing G:C to T:A mutations. Human MYH has been shown to interact physically with human proliferating cell nuclear antigen (hPCNA). Here, we report that a similar interaction between SpMYH and SpPCNA occurs in the fission yeast Schizosaccharomyces pombe. Binding of SpMYH to SpPCNA was not observed when phenylalanine 444 in the PCNA binding motif of SpMYH was replaced with alanine. The F444A mutant of SpMYH expressed in yeast cells had normal adenine glycosylase and DNA binding activities. However, expression of this mutant form of SpMYH in a SpMYHDelta cell could not reduce the mutation frequency of the cell to the normal level. Moreover, SpMYH interacted with hPCNA, and SpPCNA interacted with hMYH but not with F518A/F519A mutant hMYH containing mutations in its PCNA binding motif. Although the SpMYHDelta cells expressing hMYH had partially reduced mutation frequency, the F518A/F519A mutant hMYH could not reduce the mutation frequency of SpMYHDelta cells. Thus, the interaction between SpMYH and SpPCNA is important for SpMYH biological function in mutation avoidance.

Effects of Brussels sprouts extracts on hydrogen peroxide-induced DNA strand breaks in human lymphocytes.

Aqueous Brussels sprouts extracts inhibit oxidation of isolated DNA in vitro, possibly through scavenging oxygen radicals. We have studied the effect of preincubating human lymphocytes with aqueous extracts of raw, cooked and autolysed Brussels sprouts and the glucosinolate, sinigrin, on hydrogen peroxide-induced DNA damage, strand breaks and base oxidation, in vitro by means of the Comet assay. DNA repair enzymes endonuclease III (EndoIII) and formamidopyrimidine-DNA glycosylase (FPG) were used to examine the levels of oxidised pyrimidines and purines in DNA, respectively. Aqueous extracts of cooked and autolysed Brussels sprouts and sinigrin decreased DNA strand breaks in human lymphocytes exposed to 100 microM H2O2 for 5 min on ice, although the level of EndoIII and FPG sensitive sites was not reduced. The maximum inhibition was by 38 and 39% at concentrations of cooked and autolysed extracts of 10 microg/ml and 5 microg/ml, respectively, whereas the inhibitory effect decreased with increasing concentrations up to 100 microg/ml. The maximum inhibition by sinigrin was by 54% at 2 microg/ml. Extracts of raw Brussels sprouts or green beans had no DNA-protective effect. The results indicate that compounds, including sinigrin, in cooked and autolysed Brussels sprouts can enhance lymphocyte resistance towards H2O2-induced DNA strand breaks in vitro.

DNA glycosylase activity assay based on streptavidin paramagnetic bead substrate capture.

A method to detect DNA glycosylase activity is described. The substrate used was an oligodeoxyribonucleotide with a unique hypoxanthine base, but has general application to any DNA glycosylase or endonuclease. The oligodeoxyribonucleotide was labeled at the 5' end with 32P and at the 3' end with a biotin linkage and annealed to a complementary oligodeoxyribonucleotide. The hypoxanthine base was excised in solution using the MPG protein, a human DNA glycosylase. Following cleavage of the phosphodiester linkage by NaOH, the oligodeoxyribonucleotide was attached to streptavidin-coated, paramagnetic beads. Binding of the labeled oligodeoxyribonucleotide to the beads was indicative of the kinetics of the reaction. As a control, half of the reaction products were loaded on to a denaturing polyacrylamide gel. Comparable values for steady-state kinetic constants were obtained using both methods. This nonelectrophoretic technique is a rapid assay of DNA glycosylase activity for both purified proteins and crude extracts. This method can be directly adapted for high-throughput techniques.

Human Ogg1, a protein involved in the repair of 8-oxoguanine, is inhibited by nitric oxide.

NO-mediated inhibition of base excision DNA repair may potentiate oxidativeDNA damage in cells and could be relevant to carcinogenesis associated with chronic inflammation. Because 8-oxoguanine, a ubiquitous oxidative DNA lesion, is repaired predominantly by human 8-oxoguanine glycosylase (hOgg1), our aim was to determine whether NO directly inhibits its repair activity. Neither induction of NO-generating enzyme inducible NO synthase nor treatment with S-nitroso-N-acetyl-D-L-pencillamine altered expression of hOgg1 in a human cholangiocarcinoma cell line (KMBC). In contrast, both treatments completely inhibited activity of hOgg1 immunoprecipitated from KMBC cells overexpressing hOgg1 and in a cell-free system. Both NO and peroxynitrite were capable of inhibiting hOgg1 activity. Inhibition of hOgg1 protein was characterized by formation of S-nitrosothiol adducts and loss/ejection of zinc ions. Our data indicate that NO, an inflammatory mediator, directly inhibits a key base excision repair enzyme (hOgg1) responsible for base excision repair of 8-oxoguanine. These data support the concept that NO-mediated inhibition of DNA contributes to the mutagenic environment of chronic inflammation.