Complete nucleotide sequence analysis and identification of 7-cyano-7-deazaguanine (PreQ0) biosynthesis-related genes in the novel Bacillus subtilis-infecting Siphoviridae family phage BSP7.

In this study, bacteriophage BSP7, a novel Bacillus subtilis-infecting member of the family Siphoviridae, was isolated from a Korean soybean-based fermented food, Deonjang, using B. subtilis ATCC 21336 as a host. The genome is 55,455 bp long with 39.92% G+C content. A total of 70 ORFs with no tRNA were detected in the genome. A distinct feature of the BSP7 genome among B. subtilis-infecting Siphoviridae family phages is the presence of putative ORFs related to biosynthesis of 7-cyano-7-deazaguanine (PreQ0), a precursor of queuosine and archaeosine biosynthesis. Bioinformatic analysis revealed that the genome of BSP7 does not exhibit any significant similarities to other phages with sequences in the NCBI database. A comparative genomic analysis also confirmed the uniqueness of BSP7 within the family Siphoviridae.

Analysis of DNA Adducts and Mutagenic Potency and Specificity in Rats Exposed to Acrylonitrile.

Acrylonitrile (ACN), which is a widely used industrial chemical, induces cancers in multiple organs/tissues of rats by unresolved mechanisms. For this report, evidence for ACN-induced direct/indirect DNA damage and mutagenesis was investigated by assessing the ability of ACN, or its reactive metabolite, 2-cyanoethylene oxide (CEO), to bind to DNA in vitro, to form select DNA adducts [N7-(2'-oxoethyl)guanine, N2,3-ethenoguanine, 1,N6-ethenodeoxyadenosine, and 3,N4-ethenodeoxycytidine] in vitro and/or in vivo, and to perturb the frequency and spectra of mutations in the hypoxanthine-guanine phosphoribosyltransferase (Hprt) gene in rats exposed to ACN in drinking water. Adducts and frequencies and spectra of Hprt mutations were analyzed using published methods. Treatment of DNA from human TK6 lymphoblastoid cells with [2,3-14C]-CEO produced dose-dependent binding of 14C-CEO equivalents, and treatment of DNA from control rat brain/liver with CEO induced dose-related formation of N7-(2'-oxoethyl)guanine. No etheno-DNA adducts were detected in target tissues (brain and forestomach) or nontarget tissues (liver and spleen) in rats exposed to 0, 3, 10, 33, 100, or 300 ppm ACN for up to 105 days or to 0 or 500 ppm ACN for ∼15 months; whereas N7-(2'-oxoethyl)guanine was consistently measured at nonsignificant concentrations near the assay detection limit only in liver of animals exposed to 300 or 500 ppm ACN for ≥2 weeks. Significant dose-related increases in Hprt mutant frequencies occurred in T-lymphocytes from spleens of rats exposed to 33-500 ppm ACN for 4 weeks. Comparisons of "mutagenic potency estimates" for control rats versus rats exposed to 500 ppm ACN for 4 weeks to analogous data from rats/mice treated at a similar age with N-ethyl-N-nitrosourea or 1,3-butadiene suggest that ACN has relatively limited mutagenic effects in rats. Considerable overlap between the sites and types of mutations in ACN-exposed rats and butadiene-exposed rats/mice, but not controls, provides evidence that the carcinogenicity of these epoxide-forming chemicals involves corresponding mutagenic mechanisms.

Guanine Radicals Generated in Telomeric G-Quadruplexes by Direct Absorption of Low-Energy UV Photons: Effect of Potassium Ions.

The study deals with the primary species, ejected electrons, and guanine radicals, leading to oxidative damage, that is generated in four-stranded DNA structures (guanine quadruplexes) following photo-ionization by low-energy UV radiation. Performed by nanosecond transient absorption spectroscopy with 266 nm excitation, it focusses on quadruplexes formed by folding of GGG(TTAGGG)3 single strands in the presence of K+ ions, TEL21/K+. The quantum yield for one-photon ionization (9.4 × 10-3) was found to be twice as high as that reported previously for TEL21/Na+. The overall population of guanine radicals decayed faster, their half times being, respectively, 1.4 and 6.7 ms. Deprotonation of radical cations extended over four orders of magnitude of time; the faster step, concerning 40% of their population, was completed within 500 ns. A reaction intermediate, issued from radicals, whose absorption spectrum peaked around 390 nm, was detected.

Strand breaks and chemical modification of intracellular DNA induced by cold atmospheric pressure plasma irradiation.

DNA damage in the A549 human lung cancer cell line treated with cold plasma irradiation was investigated. We confirmed that cold atmospheric plasma generated reactive oxygen and nitrogen species (RONS) in a liquid, and the intracellular RONS level was increased in plasma-irradiated cells. However, a notable decrease in cell viability was not observed 24 hours after plasma irradiation. Because RONS induce oxidative damage in cells, strand breaks and chemical modification of DNA in the cancer cells were investigated. We found that 8-oxoguanine (8-oxoG) formation as well as DNA strand breaks, which have been thoroughly investigated, were induced by plasma irradiation. In addition, up-regulation of 8-oxoG repair enzyme was observed after plasma irradiation.

Interindividual Differences in DNA Adduct Formation and Detoxification of 1,3-Butadiene-Derived Epoxide in Human HapMap Cell Lines.

Smoking-induced lung cancer is a major cause of cancer mortality in the US and worldwide. While 11-24% of smokers will develop lung cancer, risk varies among individuals and ethnic/racial groups. Specifically, African American and Native Hawaiian cigarette smokers are more likely to get lung cancer as compared to Caucasians, Japanese Americans, and Latinos. It is important to identify smokers who are at the greatest risk of developing lung cancer as they should be candidates for smoking cessation and chemopreventive intervention programs. Among 60+ tobacco smoke carcinogens, 1,3-butadiene (BD) is one of the most potent and abundant (20-75 µg per cigarette in mainstream smoke and 205-361 µg per cigarette in side stream smoke). BD is metabolically activated to 3,4-epoxy-1-butene (EB), which can be detoxified by glutathione S-transferase theta 1 (GSTT1)-mediated conjugation with glutathione, or can react with DNA to form 7-(1-hydroxy-3-buten-2-yl)guanine (EB-GII) adducts. In the present study, we employed EBV-transformed human lymphoblastoid cell lines (HapMap cells) with known GSTT1 genotypes to examine the influence of the GSTT1 gene on interindividual variability in butadiene metabolism, DNA adduct formation/repair, and biological outcomes (apoptosis). We found that GSTT1- HapMap cells treated with EB in culture produced lower levels of glutathione conjugates and were more susceptible to apoptosis but had similar numbers of EB-GII adducts as GSTT1+ cells. Our results suggest that GSTT1 can influence an individual's susceptibility to butadiene-derived epoxides.

Biosynthesis and half-life of MBX-2168-triphosphate in herpes virus-infected cells.

The third generation of methylenecyclopropane nucleoside analogs (MCPNAs) elicit an anti-viral effect against all three sub-classes of herpes viruses without inducing cytotoxicity in vitro. It has been previously established that the mechanism of action of MCPNAs is similar to that of ganciclovir (GCV) or acyclovir (ACV). However, the activation of MBX-2168, a third generation MCPNA, involves additional and unique enzymatic steps and this process has not been examined in virus-infected cells. To that end, herpes virus-infected cells were incubated with MBX-2168, synguanol, GCV, or ACV. Incubation of HCMV-infected cells with five times the EC50 of MBX-2168 (4.0 µM), synguanol (10.5 µM), or GCV (25 µM) resulted in a time-dependent increase in triphosphate accumulation reaching a maximum of 48.1 ± 5.5, 45.5 ± 2.5, and 42.6 ± 3.7 pmol/106 cells at 120 h, respectively. Additionally, half-lives of these compounds were similar in HCMV-infected cells (GCV-TP = 25.5 ± 2.7 h; MBX-2168-TP/synguanol-TP = 23.0 ± 1.4 h). HSV-1-infected cells incubated with five times the EC50 of MBX-2168 (33.5 µM) or ACV (5.0 µM) demonstrated a time-dependent increase in triphosphate levels reaching a maximum of 12.3 ± 1.5 and 11.6 ± 0.7 pmol/106 cells at 24 h, respectively. ACV-TP and MBX-2168-TP also had similar half-lives under these conditions (27.3 ± 4.8 h and 22.2 ± 2.2 h, respectively). We therefore conclude that although MBX-2168 does not follow the classical route of nucleoside analog activation, the metabolic profile of MBX-2168 is similar to other nucleoside analogs such as GCV and ACV that do.

A LONELY GUY protein of Bordetella pertussis with unique features is related to oxidative stress.

The Gram-negative bacterium B. pertussis is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. Therefore, we focused our attention on BP1253, a predicted exported protein from B. pertussis erroneously classified as lysine decarboxylase. We showed that BP1253 shares the highly conserved motif PGGxGTxxE and the key catalytic amino-acid residues with newly structurally characterized "LONELY GUY" (LOG) proteins. Biochemical studies have confirmed that this protein functions as a cytokinin-activating enzyme since it cleaves the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases, which are the active form of plant hormones called cytokinins. Remarkably, BP1253 selectively binds monophosphate nucleotides such as AMP, GMP and CMP, showing a wider variety in binding capacity compared to other LOGs. Cytokinin production studies performed with B. pertussis have revealed 6-O-methylguanine to be the physiological product of BP1253 in agreement with the higher activity of the enzyme towards GMP. 6-O-methylguanine is likely to be responsible for the increased sensitivity of B. pertussis to oxidative stress. Although BP1253 has a primary sequence resembling the hexameric type-II LOGs, the dimeric state and the presence of specific amino-acids suggests that BP1253 can be classified as a novel type-II LOG. The discovery of a LOG along with its product 6-O-methylguanine in the human pathogen B. pertussis may lead to the discovery of unexplored functions of LOGs, broadening their role beyond plants.

Targeted and Persistent 8-Oxoguanine Base Damage at Telomeres Promotes Telomere Loss and Crisis.

Telomeres are essential for genome stability. Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere shortening. Although telomeres are hypersensitive to ROS-mediated 8-oxoguanine (8-oxoG) formation, the biological effect of this common lesion at telomeres is poorly understood because ROS have pleiotropic effects. Here we developed a chemoptogenetic tool that selectively produces 8-oxoG only at telomeres. Acute telomeric 8-oxoG formation increased telomere fragility in cells lacking OGG1, the enzyme that removes 8-oxoG, but did not compromise cell survival. However, chronic telomeric 8-oxoG induction over time shortens telomeres and impairs cell growth. Accumulation of telomeric 8-oxoG in chronically exposed OGG1-deficient cells triggers replication stress, as evidenced by mitotic DNA synthesis at telomeres, and significantly increases telomere losses. These losses generate chromosome fusions, leading to chromatin bridges and micronucleus formation upon cell division. By confining base damage to the telomeres, we show that telomeric 8-oxoG accumulation directly drives telomere crisis.

The Bacterial [Fe]-Hydrogenase Paralog HmdII Uses Tetrahydrofolate Derivatives as Substrates.

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H4 MPT+ ) with H2 . H4 MPT is a C1-carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA-G proteins. The latter are required for the biosynthesis of the prosthetic group of Hmd, the iron-guanylylpyridinol (FeGP) cofactor. This finding is intriguing because Hmd and HmdII strictly use H4 MPT derivatives that are absent in most bacteria. We identified the presence of the FeGP cofactor in D. thermolithotrophum. The bacterial HmdII reconstituted with the FeGP cofactor catalyzed the hydrogenation of derivatives of tetrahydrofolate, the bacterial C1-carrier, albeit with low enzymatic activities. The crystal structures show how Hmd recognizes tetrahydrofolate derivatives. These findings have an impact on future biotechnology by identifying a bacterial Hmd paralog.

New nucleoside hydrolase with transribosylation activity from Agromyces sp. MM-1 and its application for enzymatic synthesis of 2'-O-methylribonucleosides.

Microorganisms were screened for transribosylation activity between 2'-O-methyluridine (2'-OMe-UR) and nucleobases, for the purpose of developing a biotransformation process to synthesize 2'-O-methylribonucleosides (2'-OMe-NRs), which are raw materials for nucleic acid drugs. An actinomycete, Agromyces sp. MM-1 was found to produce 2'-O-methyladenosine (2'-OMe-AR) when whole cells were used in a reaction mixture containing 2'-OMe-UR and adenine. The enzyme responsible for the transribosylation was partially purified from Agromyces sp. MM-1 cells through a six-step separation procedure, and identified as a nucleoside hydrolase family enzyme termed AgNH. AgNH was a bi-functional enzyme catalyzing both hydrolysis towards 2'-OMe-NRs and transribosylation between 2'-OMe-UR and various nucleobases as well as adenine. In the hydrolysis reaction, AgNH preferred guanosine analogues as its substrates. In the transribosylation reaction, AgNH showed strong activity towards 6-chloroguanine, with 25-fold relative activity when adenine was used as the acceptor substrate. The transribosylation reaction product from 2'-OMe-UR and 6-chloroguanine was determined to 2'-O-methyl-6-chloroguanosine (2'-OMe-6ClGR). Under the optimal conditions, the maximum molar yield of 2'-OMe-6ClGR reached 2.3% in a 293-h reaction, corresponding to 440 mg/L.

Nitrative DNA damage in cultured macrophages exposed to indium oxide.

OBJECTIVES: Indium compounds are used in manufacturing displays of mobile phones and televisions. However, these materials cause interstitial pneumonia in exposed workers. Animal experiments demonstrated that indium compounds caused lung cancer. Chronic inflammation is considered to play a role in lung carcinogenesis and fibrosis induced by particulate matters. 8-Nitroguanine (8-nitroG) is a mutagenic DNA lesion formed during inflammation and may participate in carcinogenesis. To clarify the mechanism of carcinogenesis, we examined 8-nitroG formation in indium-exposed cultured cells. METHODS: We treated RAW 264.7 mouse macrophages with indium oxide (In2O3) nanoparticles (primary diameter: 30-50 nm), and performed fluorescent immunocytochemistry to detect 8-nitroG. The extent of 8-nitroG formation was evaluated by quantitative image analysis. We measured the amount of nitric oxide (NO) in the culture supernatant of In2O3-treated cells by the Griess method. We also examined the effects of inhibitors of inducible NO synthase (iNOS) and endocytosis on In2O3-induced 8-nitroG formation. RESULTS: In2O3 significantly increased the intensity of 8-nitroG formation in RAW 264.7 cells in a dose-dependent manner. In2O3-induced 8-nitroG formation was observed at 2 h and further increased at 4 h, and the amount of NO released from In2O3-exposed cells was significantly increased at 2-4 h compared with the control. 8-NitroG formation was suppressed by 1400W (an iNOS inhibitor), methyl-ß-cyclodextrin and monodansylcadaverine (inhibitors of caveolae- and clathrin-mediated endocytosis, respectively). CONCLUSIONS: These results suggest that endocytosis and NO generation participate in indium-induced 8-nitroG formation. NO released from indium-exposed inflammatory cells may induce DNA damage in adjacent lung epithelial cells and contribute to carcinogenesis.

Preclinical activity of FF-10501-01, a novel inosine-5'-monophosphate dehydrogenase inhibitor, in acute myeloid leukemia.

BACKGROUND: FF-10501-01 is a selective inosine monophosphate dehydrogenase (IMPDH) inhibitor that has shown activity in cancer cell lines. We studied whether FF-10501-01 is effective in targeting a variety of hypomethylating agent (HMA)-sensitive and -resistant acute myelogenous leukemia (AML) cell lines. METHODS: We treated multiple cell lines (including HMA-resistant cells) with FF-10501-01 and analyzed proliferation, apoptosis, and cell cycle status. We also assessed HMA-FF-10501-01 combinations and the ability of extracellular guanosine to rescue cell proliferation in FF-10501-01-treated cells. We performed high-performance liquid chromatography (HPLC) to study guanine nucleotide levels in treated and untreated cells. Finally, we studied the effects of FF-10501-01 in fresh peripheral blood cells taken from AML patients. RESULTS: FF-10501-01 showed a strong dose-dependent effect on proliferation and induced apoptosis at approximately 30µM. The effects of FF-10501-01 treatment on cell cycle status were variable, with no statistically significant trends. Guanosine rescued proliferation in FF-10501-01-treated cells, and HPLC results showed significant decreases in phosphorylated guanosine levels in MOLM13 cells. FF-10501-01 effectively reduced proliferation at concentrations of 300µM and above in 3 primary AML samples. CONCLUSIONS: FF-10501-01 effectively induces AML cell death and reduces AML peripheral blood cell proliferation by targeting guanine nucleotide biosynthesis regardless of HMA resistance status.

Nitrative DNA damage induced by carbon-black nanoparticles in macrophages and lung epithelial cells.

Carbon black (CB) is a nanomaterial used mainly in rubber products. Exposure to CB by inhalation causes malignant lung tumors in experimental animals. CB inhalation may cause chronic inflammation in the respiratory system, leading to carcinogenesis, but the mechanism remains unclear. Reactive oxygen and nitrogen species are generated from inflammatory and epithelial cells under inflammatory conditions, and resulting DNA damage may contribute to carcinogenesis. In this study, we performed immunocytochemistry to determine whether CB exposure induces formation of 8-nitroguanine (8-nitroG), a nitrative DNA lesion formed under inflammatory conditions, in RAW 264.7 macrophage and A549 lung epithelial cells. We compared the DNA-damaging effects of CB particles with primary diameter 56nm (CB56) and 95nm (CB95). Both types of CB induced 8-nitroG formation, mainly in the nucleus of RAW 264.7 and A549 cells, and CB95 tended to induce more 8-nitroG formation than did CB56. Flow cytometry revealed that CB95 generated larger amount of reactive oxygen species than did CB56 in RAW 264.7 cells. The Griess method showed that CB95 produced significantly larger amount of nitric oxide (NO) than did CB56. Flow cytometry showed that CB95 was more efficiently internalized into the cells than was CB56. The cellular uptake of CB and 8-nitroG formation in RAW 264.7 cells were reduced by monodansylcadaverine, an inhibitor of clathrin-mediated endocytosis, and by siRNA for Ctlc (clathrin heavy chain) gene. CB induces nitrative DNA damage in cultured cells, and clathrin-mediated endocytosis is involved, at least in part.

Dihydromethysticin (DHM) Blocks Tobacco Carcinogen 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-Induced O6-Methylguanine in a Manner Independent of the Aryl Hydrocarbon Receptor (AhR) Pathway in C57BL/6 Female Mice.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a key carcinogen responsible for tobacco smoke-induced lung carcinogenesis. Among the types of DNA damage caused by NNK and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), O6-methylguanine (O6-mG) is likely the most carcinogen in A/J mice. Results of our previous studies showed that levels of O6-mG and other types of NNAL-derived DNA damage were preferentially reduced in the lung of female A/J mice upon dietary treatment with dihydromethysticin (DHM), a promising lung cancer chemopreventive agent from kava. Such a differential blockage may be mediated via an increased level of NNAL glucuronidation, thereby leading to its detoxification. The potential of the aryl hydrocarbon receptor (AhR) as an upstream target of DHM mediating these events was evaluated herein using Ahr+/- and Ahr-/- C57BL/6 female mice because DHM was reported as an AhR agonist. DHM (0.05, 0.2, and 1.0 mg/g of diet) and dihydrokavain (DHK, an inactive analogue, 1.0 mg/g of diet) were given to mice for 7 days, followed by a single intraperitoneal dose of NNK at 100 mg/kg of body weight. The effects of DHM on the amount of O6-mG in the lung, on the urinary ratio of glucuronidated NNAL (NNAL-Gluc) and free NNAL, and on CYP1A1/2 activity in the liver microsomes were analyzed. As observed in A/J mice, DHM treatment significantly and dose-dependently reduced the level of O6-mG in the target lung tissue, but there were no significant differences in O6-mG reduction between mice from Ahr+/- and Ahr-/- backgrounds. Similarly, in both strains, DHM at 1 mg/g of diet significantly increased the urinary ratio of NNAL-Gluc to free NNAL and CYP1A1/2 enzymatic activity in liver with no changes detected at lower DHM dosages. Because none of these effects of DHM were dependent on Ahr status, AhR clearly is not the upstream target for DHM.

De Novo Guanine Biosynthesis but Not the Riboswitch-Regulated Purine Salvage Pathway Is Required for Staphylococcus aureus Infection In Vivo.

UNLABELLED: De novo guanine biosynthesis is an evolutionarily conserved pathway that creates sufficient nucleotides to support DNA replication, transcription, and translation. Bacteria can also salvage nutrients from the environment to supplement the de novo pathway, but the relative importance of either pathway during Staphylococcus aureus infection is not known. In S. aureus, genes important for both de novo and salvage pathways are regulated by a guanine riboswitch. Bacterial riboswitches have attracted attention as a novel class of antibacterial drug targets because they have high affinity for small molecules, are absent in humans, and regulate the expression of multiple genes, including those essential for cell viability. Genetic and biophysical methods confirm the existence of a bona fide guanine riboswitch upstream of an operon encoding xanthine phosphoribosyltransferase (xpt), xanthine permease (pbuX), inosine-5'-monophosphate dehydrogenase (guaB), and GMP synthetase (guaA) that represses the expression of these genes in response to guanine. We found that S. aureus guaB and guaA are also transcribed independently of riboswitch control by alternative promoter elements. Deletion of xpt-pbuX-guaB-guaA genes resulted in guanine auxotrophy, failure to grow in human serum, profound abnormalities in cell morphology, and avirulence in mouse infection models, whereas deletion of the purine salvage genes xpt-pbuX had none of these effects. Disruption of guaB or guaA recapitulates the xpt-pbuX-guaB-guaA deletion in vivo In total, the data demonstrate that targeting the guanine riboswitch alone is insufficient to treat S. aureus infections but that inhibition of guaA or guaB could have therapeutic utility. IMPORTANCE: De novo guanine biosynthesis and purine salvage genes were reported to be regulated by a guanine riboswitch in Staphylococcus aureus We demonstrate here that this is not true, because alternative promoter elements that uncouple the de novo pathway from riboswitch regulation were identified. We found that in animal models of infection, the purine salvage pathway is insufficient for S. aureus survival in the absence of de novo guanine biosynthesis. These data suggest targeting the de novo guanine biosynthesis pathway may have therapeutic utility in the treatment of S. aureus infections.

The Basics of Bacteriuria: Strategies of Microbes for Persistence in Urine.

Bacteriuria, the presence of bacteria in urine, is associated with asymptomatic, as well as symptomatic, urinary tract infection (UTI). Bacteriuria underpins some of the dynamics of microbial colonization of the urinary tract, and probably impacts the progression and persistence of infection in some individuals. Recent molecular discoveries in vitro have elucidated how some key bacterial traits can enable organisms to survive and grow in human urine as a means of microbial fitness adaptation for UTI. Several microbial characteristics that confer bacteruric potential have been identified including de novo synthesis of guanine, relative resistance to D-serine, and catabolism of malic acid. Microbial characteristics such as these are increasingly being defined through the use of synthetic human urine (SHU) in vitro as a model to mimic the in vivo environment that bacteria encounter in the bladder. There is considerable variation in the SHU model systems that have been used to study bacteriuria to date, and this influences the utility of these models. In this review, we discuss recent advances in our understanding of bacteruric potential with a focus on the specific mechanisms underlying traits that promote the growth of bacteria in urine. We also review the application of SHU in research studies modeling UTI and discuss the chemical makeup, and benefits and limitations that are encountered in utilizing SHU to study bacterial growth in urine in vitro.

Peroxynitrite modified DNA presents better epitopes for anti-DNA autoantibodies in diabetes type 1 patients.

Peroxynitrite (ONOO(-)), formed by the reaction between nitric oxide (NO) and superoxide (O2(-)), has been implicated in the etiology of numerous disease processes. Peroxynitrite interacts with DNA via direct oxidative reactions or via indirect radical-mediated mechanism. It can inflict both oxidative and nitrosative damages on DNA bases, generating abasic sites, resulting in the single strand breaks. Plasmid pUC 18 isolated from Escherichiacoli was modified with peroxynitrite, generated by quenched flow process. Modifications incurred in plasmid DNA were characterized by ultraviolet and fluorescence spectroscopy, circular dichroism, HPLC and melting temperature studies. Binding characteristics and specificity of antibodies from diabetes patients were analyzed by direct binding and inhibition ELISA. Peroxynitrite modification of pUC 18 plasmid resulted in the formation of strand breaks and base modification. The major compound formed when peroxynitrite reacted with DNA was 8-nitroguanine, a specific marker for peroxynitrite induced DNA damage in inflamed tissues. The concentration of 8-nitroguanine was found to be 3.8 µM. Sera from diabetes type 1 patients from different age groups were studied for their binding to native and peroxynitrite modified plasmid. Direct binding and competitive-inhibition ELISA results showed higher recognition of peroxynitrite modified plasmid, as compared to the native form, by auto-antibodies present in diabetes patients. The preferential recognition of modified plasmid by diabetes autoantibodies was further reiterated by gel shift assay. Experimentally induced anti-peroxynitrite-modified plasmid IgG was used as a probe to detect nitrosative lesions in the DNA isolated from diabetes patients.

Mitochondrial 8-oxoguanine glycosylase decreases mitochondrial fragmentation and improves mitochondrial function in H9C2 cells under oxidative stress conditions.

The mitochondrial DNA base modification 8-hydroxy 2'-deoxyguanine (8-OHdG) is one of the most common DNA lesions induced by reactive oxygen species (ROS) and is considered an index of DNA damage. High levels of mitochondrial 8-OHdG have been correlated with increased mutation, deletion, and loss of mitochondrial (mt) DNA, as well as apoptosis. 8-Oxoguanosine DNA glycosylase-1 (OGG1) recognizes and removes 8-OHdG to prevent further DNA damage. We evaluated the effects of OGG1 on mtDNA damage, mitochondrial function, and apoptotic events induced by oxidative stress using H9C2 cardiac cells treated with menadione and transduced with either Adv-Ogg1 or Adv-Control (empty vector). The levels of mtDNA 8-OHdG and the presence of apurinic/apyrimidinic (AP) sites were decreased by 30% and 35%, respectively, in Adv-Ogg1 transduced cells (P < 0.0001 and P < 0.005, respectively). In addition, the expression of base excision repair (BER) pathway members APE1 and DNA polymerase γ was upregulated by Adv-Ogg1 transduction. Cells overexpressing Ogg1 had increased membrane potential (P < 0.05) and decreased mitochondrial fragmentation (P < 0.005). The mtDNA content was found to be higher in cells with increased OGG1 (P < 0.005). The protein levels of fission and apoptotic factors such as DRP1, FIS1, cytoplasmic cytochrome c, activated caspase-3, and activated caspase-9 were lower in Adv-Ogg1 transduced cells. These observations suggest that Ogg1 overexpression may be an important mechanism to protect cardiac cells against oxidative stress damage.

Microwhip scorpions (Palpigradi) feed on heterotrophic cyanobacteria in Slovak caves--a curiosity among Arachnida.

To date, only morphological and anatomical descriptions of microwhip scorpions (Arachnida: Palpigradi) have been published. This very rare group is enigmatic not only in its relationships to other arachnids, but especially due to the fact that these animals dwell only underground (in caves, soil, and interstitial spaces). We observed the curious feeding habit of the microwhip scorpion Eukoenenia spelaea over the course of one year in Ardovská Cave, located in Slovakia's Karst region. We chose histology as our methodology in studying 17 specimens and based it upon Masson's triple staining, fluorescent light and confocal microscopy. Single-celled cyanobacteria (blue-green algae) were conspicuously predominant in the gut of all studied palpigrades. Digestibility of the consumed cyanobacteria was supported by the presence of guanine crystals, glycogen deposits and haemocytes inside the palpigrade body. Cyanobacteria, the oldest cellular organisms on Earth, are very resistant to severe conditions in caves, including even darkness. Therefore, the cyanobacteria are able to survive in dark caves as nearly heterotrophic organisms and are consumed by cave palpigrades. Such feeding habit is extraordinary within the almost wholly predacious orders of the class Arachnida, and particularly so due to the type of food observed.

Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis.

Nitrative and oxidative DNA damage plays an important role in inflammation-related carcinogenesis. Chronic inflammation such as parasite infection and primary sclerosing cholangitis can be an etiological factor of cholangiocarcinoma. Using a proteomic approach and double-fluorescent staining, we identified high expression and colocalization of albumin and cytokeratin-19 in liver fluke-associated cholangiocarcinoma tissues, compared with normal livers from cholangiocarcinoma patients and cadaveric donors, respectively. Albumin was detected not only in cells of hyperplastic bile ducts and cholangiocarcinoma, but also in liver stem/progenitor cell origin, such as canal of Hering, ductules, and ductular reactions, suggesting the involvement of stem/progenitor cells in cholangiocarcinoma development. To clarify the involvement of liver stem/progenitor cells in cholangiocarcinoma, we examined several stem/progenitor cell markers (CD133, CD44, OV6, and Oct3/4) in cholangiocarcinoma tissues analyzed by immunohistochemical staining, and measured 8-oxodG levels by using HPLC-ECD as an inflammation-related DNA lesion. In addition, a stem/progenitor cell factor Bmi1, 8-nitroguanine (formed during nitrative DNA damage), DNA damage response (DDR) proteins (phosphorylated ATM and γ-H2AX), and manganese-SOD (Mn-SOD) were analyzed by immunohistochemistry. Stem/progenitor cell markers (CD133, OV6, CD44, and Oct3/4) were positively stained in 56, 38, 47, and 56% of 34 cholangiocarcinoma cases, respectively. Quantitative analysis of 8-oxodG revealed significantly increased levels in CD133- and/or Oct3/4-positive tumor tissues compared to negative tumor tissues, as well as 8-nitroguanine formation detected by immunohistochemistry. In the cases of CD44- and/or OV6-positive tissue, no significant difference was observed. Cholangiocarcinoma patients with CD133- and/or Oct3/4-positive tumor tissues showed significantly lower expression of Mn-SOD and higher DDR protein, γ-H2AX. Moreover, CD133- and/or Oct3/4-positive cholangiocarcinoma patients had significant associations with tumor histology types, tumor stage, and poor prognoses. Our results suggest that CD133 and Oct3/4 in cholangiocarcinoma are associated with increased formation of DNA lesions and the DDR protein, which may be involved in genetic instability and lead to cholangiocarcinoma development with aggressive clinical features.