Arabidopsis thaliana Ogg1 Protein Excises 8-Hydroxyguanine and 2,6-Diamino-4-hydroxy-5-formamidopyrimidine from Oxidatively Damaged DNA Containing Multiple Lesions

A functional homologue of eukaryotic Ogg1 proteins in the model plant Arabidopsis thaliana has recently been cloned, isolated and characterized [Garcia-Ortiz, M. V., Ariza, R. R., and Roldan-Arjona, T. (2001) Plant Mol. Biol. 47, 795-804]. This enzyme (AtOgg1) exhibits a high degree of sequence similarity in several highly conserved regions with Saccharomyces cerevisiae, Drosophila melanogaster and human Ogg1 proteins. We investigated the substrate specificity and kinetics of AtOgg1 for excision of modified bases from oxidatively damaged DNA that contained multiple pyrimidine- and purine derived lesions. Two different DNA substrates prepared by exposure to ionizing radiation in aqueous solution under N2O or air were used for this purpose. Gas chromatography/isotope-dilution mass spectrometry was applied to identify and quantify modified bases in DNA samples. Of the seventeen modified bases identified in DNA samples, only 8-hydroxyguanine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine were significantly excised from both DNA substrates. This is in agreement with the substrate specificities of other eukaryotic Ogg1 proteins that had previously been studied under identical conditions. Excision depended on incubation time, enzyme concentration and substrate concentration, and followed Michaelis-Menten kinetics. A significant dependence of excision on the nature of DNA substrate was observed in accord with previous studies on other DNA glycosylases. A comparison of excision kinetics pointed to significant differences between AtOgg1 and other Ogg1 proteins. We also investigated the effect of base-pairing on the excision using double-stranded oligodeoxynucleotides that contained 8-OH-Gua paired with each of the four DNA bases. The activity of AtOgg1 was most effective on the 8-OH-Gua:C pair with some or very low activity on other pairs in agreement with the activity of other Ogg1 proteins. The results unequivocally show that AtOgg1 possesses common substrates with other eukaryotic Ogg1 proteins albeit significant differences between their excision kinetics. Oxygen-derived species such as free radicals and other oxidizing agents generate a multiplicity of lesions in DNA comprising modified bases and sugars, DNA-protein cross-links, strand breaks and base-free sites (reviewed in ref (1;2)). A variety of repair pathways exist in cells to combat DNA damage and to maintain genomic integrity (reviewed in ref (3)). The repair of modified bases in DNA of both eukaryotes and prokaryotes primarily occurs via the base-excision repair (BER)1 pathway (reviewed in ref (4)), which is conserved throughout all species. DNA glycosylases are involved in the first step of BER and remove modified bases from DNA by catalyzing the hydrolysis of the glycosidic bond between the modified base and the sugar moiety. In Escherichia coli, formamidopyrimidine DNA glycosylase (Fpg) is a DNA glycosylase/lyase that excises 8-hydroxyguanine (8-OH-Gua), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) and 4,6-diamino-5-formamidopyrimidine (FapyAde) from DNA and processes the resulting abasic site by cleaving both 3´- and 5´-phosphodiester bonds by successive β- and δ-eliminations (5-7). Fpg shows a clear preference for Cyt as the base opposite 8-OH-Gua, with 8-OH-Gua:Ade being a particularly poor substrate (8). This specificity avoids repair of 8-OH-Gua:Ade mispairs to T:A, which would cause a GC→TA transversion. The repair of 8-OH-Gua:Ade mispairs is initiated instead by MutY, a DNA glycosylase that catalyze excision of misincorporated Ade (9). Animals and yeast cells possess 8-OH-Gua DNA glycosylases that do not share significant sequence identity with bacterial Fpg proteins. The first of these Fpg analogues was identified in Saccharomyces cerevisiae and designated yOgg1 (10;11). This enzyme possesses a similar substrate specificity to Fpg and removes 8-OH-Gua and FapyGua, but not FapyAde in contrast to Fpg (12), and displays a preference for 8-OH-Gua opposite Cyt, but not Ade (10;11;13). Genes encoding proteins, which share significant sequence identity with yOgg1, have been subsequently cloned and characterized in humans and other mammals (14-20). Genome analyses revealed proteins similar to Ogg1 in Archaea but not in any bacterial species (21). Arabidopsis thaliana has become a very attractive model system for study of conserved DNA repair pathways due to the practical advantages of small size and rapid life cycle in conjunction with the recent development of powerful tools to study its genome. In addition, there are important differences between the life strategies of plants and most eukaryotes. For example, plants do not have a reserve germ line, and their gametes differentiate late in development from somatic cells. This and other differences may shed light on crucial aspects of genome-maintenance functions in eukaryotes. We recently isolated and characterized an Ogg1 orthologue in the model plant Arabidopsis thaliana (AtOgg1) (22). Our finding was of particular interest, since an 8-OH-Gua DNA glycosylase (AtMMH) encoded by a gene named AtMMH, which is an orthologue of E. coli’s gene Fpg, had previously been isolated and characterized in this model plant (23). The discovery of AtOgg1 established plants as the only organisms, where the presence of both Fpg and Ogg1 homologues exist. On the other hand, Arabidopsis is not the only example of an organism with two different enzymes for the repair of 8-OH-Gua. In Drosophila melanogaster, both the ribosomal protein S3 and an Ogg1 protein (dOgg1), which is a true orthologue of other Ogg1 proteins, possess DNA glycosylase/β-lyase activity capable of releasing 8-OH-Gua and FapyGua from damaged DNA with multiple lesions (24;25). In the case of Arabidopsis, the relative roles of AtOgg1 and AtMMH might be connected to their likely different phylogenetic origin, since the latter may be the result of a gene transfer from an ancestral chloroplast genome to the nucleus (23). Although there are putative nuclear targeting signals in the predicted amino acid sequences of AtOgg1 and AtMMH, the subcellular localization of both enzymes remains to be determined. The possibility exists that AtOgg1 removes modified bases for oxidatively damaged DNA in the nucleus, while AtMMH continues performing an essential DNA repair activity in the chloroplast genome. In the present work, we report the substrate specificity and excision kinetics of AtOgg1 using oxidatively damaged DNA containing a multiplicity of lesions. Excision by AtOgg1of modified DNA bases was studied using the technique of gas chromatography/isotope-dilution mass spectrometry (GC/IDMS). Furthermore, an oligodeoxynucleotide containing a single 8-OH-Gua residue at a defined position, which was paired with each of the four DNA bases, were used to investigate the paired-base effect on the repair activity of this protein

The evaluation of deaths due to methyl alcohol intoxication

Background: Methanol poisoning is a serious medical, social and economic problem that may cause severe illness or death. After methanol ingestion, central nervous system depression, headache, dizziness, nausea, lack of coordination, and confusion begins. Once the initial symptoms have passed, a second set of symptoms arises, 10 to 30 hours after the initial exposure to methanol, including blurring or complete loss of vision and acidosis. Methanol poisoning by ingestion is a world-wide problem, and in some regions it is connected with high morbidity and mortality. The lethal dose of methanol in humans shows pronounced individual differences ranging from 15 to 500 ml. Methods: The records of the First Specialization Board of the Council of Forensic Medicine between 2002 and 2010 were reviewed retrospectively for all methyl alcohol poisoning cases. Results: There were 383 cases recorded. 360 (94%) of total fatalities were men and 23 (6%) were women. The age range was between 17 and 89. Although patients were conscious, cooperative, oriented at first, deteriorated general health state, metabolic acidosis and neurologic sequelae with severe electrocardiographic (ECG) changes were seen in progress of time. The laboratory findings and MRI imaging method were applied to assess progress and medical treatment. Unfortunately severe acidosis, central nervous system (CNS) sequelae and a lethal outcome occurred. The methyl alcohol blood concentrations ranged from 0 to 826 mg per 100 ml. The most common macroscopic and microscopic finding was lung edema, cerebral and cerebellar hemorrhage, ischemic changes in the brain and optic neuritis. Putaminal necrosis and hemorrhage, brainstem petechial hemorrhage, myocardial acute ischemic changes, thalamic and hypothalamic hemorrhage were detected rarely. Conclusions: This is the first study to report postmortem findings, clinical reports, crime scene reports and eye witness accounts to investigate methyl alcohol poisoning cases from medico-legal point of view in Turkey. Methanol poisoning by ingestion is a world-wide problem with high morbidity and mortality. For preventing methanol deaths, both awareness and public education must be increased. © 2017, Scientific Publishers of India. All rights reserved

Interaction of Risk Factors, Comorbidities, and Comedications with Ischemia/Reperfusion Injury and Cardioprotection by Preconditioning, Postconditioning, and Remote Conditioning

Pre-, post-, and remote conditioning of the myocardium are well described adaptive responses that markedly enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and provide therapeutic paradigms for cardioprotection. Nevertheless, more than 25 years after the discovery of ischemic preconditioning, we still do not have established cardioprotective drugs on the market. Most experimental studies on cardioprotection are still undertaken in animal models, in which ischemia/reperfusion is imposed in the absence of cardiovascular risk factors. However, ischemic heart disease in humans is a complex disorder caused by, or associated with, cardiovascular risk factors and comorbidities, including hypertension, hyperlipidemia, diabetes, insulin resistance, heart failure, altered coronary circulation, and aging. These risk factors induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury per se and responses to cardioprotective interventions. Moreover, some of the medications used to treat these risk factors, including statins, nitrates, and antidiabetic drugs, may impact cardioprotection by modifying cellular signaling. The aim of this article is to review the recent evidence that cardiovascular risk factors and their medication may modify the response to cardioprotective interventions. We emphasize the critical need to take into account the presence of cardiovascular risk factors and concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple risk factors

Heart echinococcus cyst as an incidental finding: early detection might be life-saving

We present a 46-year-old female smoker who was admitted to the emergency department of our hospital due to cough with blood-tinged sputum for the last four days before admission. Using echocardiography and Multi-Detector Computed Tomography (MDCT) heart Echinococcosis was diagnosed. Echinococcosis is a severe health issue in some geographical regions of the world. Hydatid infection of the heart is rare and the clinical presentation is usually insidious but there is always the lethal hazard of cyst perforation. Early diagnosis and an integrated treatment strategy are crucial. The results of surgical treatment of heart echinococcosis are better than the conservative strategy only. Extraction of the cyst combined with chemotherapy peri or post operative aiming to decrease the recurrences, consists the lege artis method of encountering this medical entity. Surgical excision was performed and the patient had an uneventful recovery and follow up at six and twelve months

Experimental neuronal lipofuscinosis in sheep fed with Asphodelus aestivus seeds; Pathological and ultrastructural investigations

Asphodelus aestivus (A. aestivus) is a common plant in the meadows in Aydin region, Turkey. Severe neurologic syndromes accompanied by intense neurovisceral lipofuscinosis were observed in sheep exposed to A. aestivus leaves and seeds in the same region. Findings from dead sheep indicated that consumption of A. aestivus seeds resulted in neuronal lipofuscinosis. Hence, an experiment with four treatment groups was carried out to induce neuronal lipofuscinosis in sheep consuming A. aestivus seeds at different levels in the diets. Animals in treatment groups were fed with total 600 g/ daily yearling feed including 0% (Control group; animal 1 and 2, 2 years old - animal 3; 3 years old), 5% (Treatment I; animal 1 and 2; 2 years old), 10% (Treatment II; animal 3; 3 years old - animal 4; 2 years old) and 15% (Treatment III; animal 5 and 6; 2 years old) of A. aestivus seeds during the first four months of the experiment. Then they were fed with total 600 g/ daily yearling feed including 0% (Control group), 15% (Treatment I), 20% (Treatment II) and 35% (Treatment III) of A. aestivus seeds during the last two months of the experiment. At the end of the experiment, systemic necropsies of the sheep were performed. Tissue samples were collected from all organs and fixed in 10% neutral formalin. After the samples were stained with hematoxylin eosin, they were examined by light microscope. The ultrastructural sections prepared from pons and medulla oblongata were examined by electron microscope. Clinically, a general toxicity accompanied by loss of weight, transient paresis and hyperesthesia was observed in the Treatment III. The neuronal lipofuscinosis were determined particularly in neurons of the brain stem and in myenteric plexus of the intestines of sheep on the test diets