DNA Degradation Products Research Articles

Evaluation of beneficial effects of dexpanthenol on hypoxic-ischemic encephalopathy

ABSTRACT Hypoxic-ischemic encephalopathy (HIE) is a cause of serious morbidity and mortality in newborns. Dexpanthenol, which is metabolized into D-pantothenic acid, has antioxidant and other potentially therapeutic properties. We examined some effects of dexpanthenol on the brains of week-old rat pups with HIE induced by obstruction of the right carotid artery followed by keeping in 8% O2 for 2 hours. Dexpanthenol (500 mg/kg) was administered intraperitoneally to 16 of 32 pups with HIE. Protein, DNA, and lipid oxidation degradation products were assayed and hippocampal and cortical cell apoptosis and neuronal cell numbers were evaluated in stained sections. Dexpanthenol application reduced oxidative stress and inflammation. TNF-α and IL-6 cytokine levels in HIE also decreased with dexpanthenol treatment. The numbers of caspase-3 positive cells in the dentate gyrus and CA1/CA2/CA3 regions of the hippocampus was lower, and apoptosis was decreased in the dexpanthenol-treated animals. These findings suggest possible clinical applications of dexpanthenol in human HIE.

Follicular fluid redox balance and DNA damage according to PCOS phenotype.

Classification of polycystic ovary syndrome (PCOS) according to their phenotypes is important in terms of understanding which parameter has clinical and laboratory implications. This study was designed to measure the follicular fluid total oxidant capacity (TOC) and total antioxidant capacity (TAC) and DNA degradation products of 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in patients with different PCOS phenotypes undergoing In-Vitro Fertilization/Intra-Cytoplasmic Sperm Injection (IVF/ICSI). Thirty women who were diagnosed with PCOS and twenty infertile patients who did not have the clinical and laboratory findings of PCOS were included. Women carrying at least two of the three parameters below were considered to have PCOS. (1) Biochemical or clinical manifestations of hyperandrogenism (HA); (2) Ovulatory dysfunction (OD); (3) Polycystic ovarian morphology (PCOM). Patients were then classified into four different PCOS phenotypes: (1) Phenotype A is also known as classical PCOS and meets all three criteria (HA/OD/PCOM). (2) Phenotype B has two criteria, HA and OD. (3) Phenotype C consists of HA and PCOM criteria. (4) Phenotype D is the non-hyperandrogenic form and consists of OD and PCOM criteria. Antagonist protocol was used in both PCOS and control groups. During oocyte pick-up, follicular fluid of the dominant follicle was collected. TAC and TOC levels, which are redox balance markers, and 8-OHdG levels, which are DNA degradation products, were measured in follicular fluid samples (FF). Follicular fluid 8-OHdG levels of all four types of phenotypes were significantly higher than the control group. When the phenotype groups were evaluated among themselves, FF-8-OHdG levels of each group were found to be similar. Serum TOC levels of each phenotype group were found to be significantly higher than the control group. TAC levels of the patients in the control group were significantly higher than the other four phenotype groups. Oxidative stress index (OSI) values were significantly higher in all four phenotype groups compared to the control group. OSI values of phenotype B and D groups were significantly higher than phenotypes A and C. In each phenotype of PCOS, TOC and OSI increased while TAC decreased. Increased OSI leads to DNA degradation and an increase in the level of 8-OHdG. The cumulative effect of oxidative stress and DNA degradation may be the main mechanism of PCOS-related subfertility.

8-Hydroxy-2-deoxyguanosine, a product of oxidative DNA degradation, is increased in the amniotic fluid of preterm births.

8-Hydroxy-2-deoxyguanosine (8-OH-2dG) is a measurable biomarker of oxidative DNA damage. This study was designed to determine amniotic fluid 8-OH-2dG levels in healthy full-term pregnant women and preterm pregnant women. To reveal the effect of reactive oxygen species on 8-OH-2dG levels, amniotic fluid total oxidant capacity (TOS), total antioxidant capacity (TAC) and oxidative stress index (OSI) were also measured. A total of 60 patients, 35 patients with full-term pregnancy and 25 patients with preterm pregnancy, participated in the study. Labor occurring before 37 weeks of gestation was considered as spontaneous preterm birth. Amniotic fluid samples were collected from full-term patients during cesarean section or normal vaginal delivery. 8-OH-2dG concentrations in amniotic fluid samples were measured quantitatively by Enzyme-Linked Immunosorbent Assay (ELISA). Amniotic fluid total antioxidant capacity (TAC) and total oxidant capacity (TOC) was determined in amniotic samples. The amniotic fluid 8-OH-2dG levels of the preterm group were significantly higher than the full-term group (60.8±7.02 ng/mL vs. 33.6±4.11 ng/mL, p<0.01). Similarly, TOC levels of the preterm group were significantly higher than the full-term group (89.7±4.80 µmol/L vs. 54.3±6.60 µmol, p<0.02). TAC was significantly higher in the full-term group compared to the preterm group (1.87±0.10 mmol/L vs. 0.97±0.44 mmol/L, p<0.01). The OSI values of the preterm group were significantly higher than the full-term group. A negative and significant correlation was found between gestational age and amniotic fluid 8-OH-2dG levels in the full-term pregnancy group (r=-0.78, p<0.01). A negative and significant correlation was observed between TAC and amniotic fluid 8-OH-2dG levels in the full-term group (r=-0.60, p<0.02). A positive and significant correlation was also detected between TOC, OSI and amniotic fluid 8-OH-2dG levels in the full-term group. There was a negative but insignificant correlation between fetal weight and amniotic fluid 8-OH-2dG levels. The correlation analysis results of the preterm pregnancy group were similar to the full-term group. Increased reactive oxygen derivatives in preterm birth increase amniotic fluid levels of DNA degradation product 8-OH2dG and may lead to premature rupture of fetal membranes. This is the first clinical study investigating 8-OH-2dG levels in amniotic fluid of preterm birth.

Post-COVID syndrome is associated with increased extracellular purine bases and neutrophil extracellular traps in the blood plasma

Post-COVID syndrome is characterized by fatigue, reduced exercise tolerance, muscle and joint pain, and psychoemotional disorders. In the development of a generalized body response in a viral infection, abnormal defense responses are of great importance. We studied neutrophils, neutrophil extracellular traps (NETs), DNA degradation products (purine nitrogenous bases, PNBs), and traditional biochemical parameters.Aim. To determine biochemical parameters and the number of NETs and PNBs in the peripheral blood of patients with post-COVID syndrome.Materials and methods. The study included outpatients (n = 21) aged 18–59 years (36 [27 ÷ 50]). The control group consisted of 20 individuals aged 18–59 years (38.5 [29 ÷ 51.5]) without a past medical history of the coronavirus infection. All patients underwent a physical examination, their medical history was assessed, and the level of NETs and PNBs in the venous blood was determined.Results. 11 patients had a mild form of the disease in their past medical history, 7 – moderate, and 3 – severe. The most common symptoms in the patients were fatigue, headache, epigastric pain, dizziness, and joint pain. Hair loss and dyspnea were less common. The concentration of NETs and PNBs was higher in the patients with post-COVID syndrome than in the control group (p &lt; 0.05). We detected NETs in the patients with post-COVID syndrome only in the form of filamentous structures. The concentration of extracellular purine bases in the blood of the patients with post-COVID syndrome was the highest in patients with moderate and severe acute periods. In patients with a mild acute period, the concentration of PNBs was 7.38 [0.0 ÷ 60.7] mg / ml, and in patients with moderate and severe acute periods – 19.15 [0.0 ÷ 33.5] and 34.19 [3.35 ÷ 70.0] mg / ml, respectively.Conclusion. Extracellular purine bases in concentrations capable of causing secondary alteration of cells are found in the peripheral blood of patients with post-COVID syndrome. Post-COVID syndrome is accompanied by the formation of filamentous NETs in the blood of patients.

Potential Phosphorus Uptake Mechanisms in the Deep Sedimentary Biosphere

Our understanding of phosphorus (P) dynamics in the deep subseafloor environment remains limited. Here we investigate potential microbial P uptake mechanisms in oligotrophic marine sediments beneath the North Atlantic Gyre and their effects on the relative distribution of organic P compounds as a function of burial depth and changing redox conditions. We use metagenomic analyses to determine the presence of microbial functional genes pertaining to P uptake and metabolism, and solution 31P nuclear magnetic resonance spectroscopy (31P NMR) to characterize and quantify P substrates. Phosphorus compounds or compound classes identified with 31P NMR include inorganic P compounds (orthophosphate, pyrophosphate, polyphosphate), phosphonates, orthophosphate monoesters (including inositol hexakisphosphate stereoisomers) and orthophosphate diesters (including DNA and phospholipid degradation products). Some of the genes identified include genes related to phosphate transport, phosphonate and polyphosphate metabolism, as well as phosphite uptake. Our findings suggest that the deep sedimentary biosphere may have adapted to take advantage of a wide array of P substrates and could play a role in the gradual breakdown of inositol and sugar phosphates, as well as reduced P compounds and polyphosphates.

Abstract B39: A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens

Abstract Objective: We propose to utilize tumor biospecimens from patients with esophageal squamous cell carcinoma (ESCC) to identify possible genetic, molecular, and infectious determinants of this high-incidence disease in East Africa. However, laboratory technologies in East Africa are not available to support DNA and RNA extraction and genome and transcriptome sequencing from patient specimens. In preparation for establishment of a biorepository for African ESCC specimens, we compared different fixation and preservation media for transportability over long distances and success in preserving the genetic integrity and expression profiles of ESCC. Methods: Patients with a suspected diagnosis of ESCC at Muhimbili National Hospital in Dar es Salaam, Tanzania were identified and consented prior to endoscopic evaluations. For patients with endoscopic findings consistent with ESCC, tumor biopsies were obtained and stored using two different fixation and preservation media: PAXgene® Tissue Container (n=2) and RNAlater® (n=2). All specimens were shipped at room temperature from Dar es Salaam to San Francisco. DNA and RNA quantity was measured by nanodrop method and DNA was further confirmed by picogreen method, yielding measures of total DNA and RNA acquired (ug). DNA quality was measured as percent of total DNA degradation to between 200-1000 bp. RNA quality measure was determined by bioanalyzer output measure RNA Integrity Number (RIN). Results: Specimens for our first 10 patients were analyzed. Average transit time of biopsy specimens in preservative at room temperature was 6.2 days (range 3.5-9.0). Tumor specimens preserved with PAXgene® yielded a mean of 7.7 ug total DNA and 8.6 ug total RNA. Tumor specimens preserved with RNAlater® yielded a mean 1.4 ug of DNA and 8.2 ug of RNA. DNA degradation products were 0% of total with PAXgene® versus 6% of total with RNAlater®. Specimens preserved with PAXgene® yielded a mean RIN of 4, while RNAlater® yielded a mean RIN of 9. DNA and RNA quality were not associated with length of time at room temperature, up to a maximum of 9 days. Conclusion: Tissue preserved using the PAXgene® Tissue Container yielded higher DNA quantity and quality than tissue preserved in RNAlater®. RNA quantity was comparable for both mediums, but RNAlater® resulted in superior RNA quality versus PAXgene®. Both mediums allowed for flexible transport of specimens at room temperature and merit further inquiry into their potential as cost-effective methods to facilitate molecular analyses for geographically isolated diseases in Africa. Based upon our pilot data, each medium offers unique advantages. Our expanded study will continue to utilize both mediums to optimize isolation of both DNA and RNA. We will plan to present data for an enriched sample size. Funding source: National Institutes of Health, National Cancer Institute Cancer Center Administrative Supplement to Promote Cancer Prevention and Control Research in Low and Middle Income Countries, A119617, [CA-0082629]. Citation Format: Beatrice Paul Mushi, John Greer, Charles William Cahalane, II, Msiba Selekwa, Ali Mwanga, Larry Akoko, Elia Mmbaga, Eric Collisson, Katherine Van Loon. A pilot study to establish procedures for DNA and RNA isolation from African esophageal tumor specimens [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B39.

Short single-stranded DNA degradation products augment the activation of Toll-like receptor 9

Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.

Evaluation of the role of captopril on clozapine-induced cardiotoxicity and hematotoxicity in adult male albino rats

Clozapine (CLZ) is considered the most effective drug in treatment of resistant schizophrenia. However, its cardiotoxic effect has raised concerns about its safety. Captopril is a well-known angiotensin-converting enzyme inhibitor with unique antioxidant properties. The aim of this study was to investigate the protective effect of captopril against CLZ-induced myocarditis, and since both drugs have hematotoxic effects, this study aimed to clarify the effect of their combined use on the bone marrow. The study was conducted for 4 weeks on 50 adult male albino rats divided into five groups: group I (negative control), group II (positive control), group III treated with captopril 5 mg/kg/day, group IV treated with CLZ 25 mg/kg/day, and group V treated with captopril (5 mg/kg) 1 hour before CLZ (25 mg/kg/day). CLZ group showed a significant increase in serum troponin I, marked histopathological changes, and immunohistochemical staining of DNA degradation product 8-hydroxy-2-deoxy guanosine (8-OHdG). It significantly increased malondialdehyde level and decreased glutathione peroxidase. Captopril coadministration decreased the histopathological hallmarks and biochemical marker of myocarditis and attenuated CLZ effects on the oxidative stress parameters and 8-OHdG, suggesting its protective action against CLZ-induced myocarditis. Complete blood count and bone marrow evaluation was normal indicating that captopril, in the protective dose given, didn’t increase the risk of CLZ-induced hematotoxicity

CUPRAC colorimetric and electroanalytical methods determining antioxidant activity based on prevention of oxidative DNA damage

An unbalanced excess of oxygen/nitrogen species (ROS/RNS) can give oxidative hazard to DNA and other biomacromolecules under oxidative stress conditions. While the ‘comet’ assay for measuring DNA damage is neither specific nor practical, monitoring oxidative changes on individual DNA bases and other oxidation products needs highly specialized equipment and operators. Thus, we developed a modified CUPRAC (cupric ion reducing antioxidant capacity) colorimetric method to determine the average total damage on DNA produced by Fenton oxidation, taking advantage of the fact that the degradation products of DNA but not the original macromolecule is CUPRAC−responsive. The DNA−protective effects of water-soluble antioxidants were used to devise a novel antioxidant activity assay, considered to be physiologically more realistic than those using artificial probes. Our method, based on the measurement of DNA oxidative products with CUPRAC colorimetry proved to be 2 orders-of-magnitude more sensitive than the widely used TBARS (thiobarbituric acid-reactive substances) colorimetric assay used as reference. Additionally, the DNA damage was electrochemically investigated using pencil graphite electrodes (PGEs) as DNA sensor platform in combination with differential pulse voltammetry (DPV). The interaction of the radical species with DNA in the absence/presence of antioxidants was detected according to the changes in guanine oxidation signal.

One-pot process of 2′-deoxyguanylic acid catalyzed by a multi-enzyme system

2′-Deoxyguanylic acid (deoxyguanosine-5′-monophosphate, dGMP) is a substance required by living cells that is used extensively in reagents, fine chemicals and other industrial fields. Traditionally, dGMP is separated from DNA degradation products, which is low-yielding and time-consuming. Herein, we investigated a novel, one-pot multi-enzymatic cascade reaction to produce dGMP. This reaction involved purine nucleoside phosphorylase (PNPase) and acetate kinase (ACKase) from Escherichia coli, N-deoxyribosytransferase II (NDT-II) from Lactobacillus delbrueckii and deoxyguanosine kinase (dGKase) from Bacillus subtilis. During the reaction, the initial guanosine substrate was cleaved into guanine and ribose-1-phosphate by PNPase. Then, deoxyguanosine (dGR) was subsequently produced from a reaction between guanine and thymidine catalysed by NDT-II. Finally, the intermediate dGR was phosphorylated to dGMP by dGKase and a cytidine triphosphate (CTP) regeneration system that utilised acetyl phosphate via ACKase. A very small amount of CTP was added because CTP regeneration was efficient to transfer a phosphate group from acetyl phosphate to dGR. After 12 h of incubation, a maximal dGMP yield of up to 76% was obtained based on the addition of 5 mM guanosine and 5 mM thymidine.

Protective effect of captopril against clozapine-induced myocarditis in rats: Role of oxidative stress, proinflammatory cytokines and DNA damage

Clozapine (CLZ) is the most effective therapeutic alternative in the treatment of resistant schizophrenia. However, the cardiotoxicity of CLZ, particularly in young patients, has raised concerns about its safety. Captopril is a well-known angiotensin-converting enzyme inhibitor with antioxidant properties effective in treating hypertension and heart failure. The aim of this study was to investigate the protective effect of captopril against clozapine-induced myocarditis in rats and the possible mechanisms behind this effect. The effect of captopril treatment [5 or 10mg/kg/d, injected intraperitoneally (i.p.) for 21days] on the cardiotoxic effect of coadministered CLZ (25mg/kg/d, i.p.) was assessed. Myocarditis was assessed histopathologically, immunohistochemically and biochemically. Frozen heart specimens were used to determine the amount of lipid peroxides product (MDA), nitric oxide (NO), reduced glutathione (GSH), glutathione peroxidase (GSH-Px) activity, proinflammatory cytokines (TNF-α and IL-10) and DNA degradation product(8-OHdG). Coadministration of captopril with the tested doses of CLZ decreased the histological hallmarks and biochemical markers (CK-MP and LDH) of myocarditis. In addition, captopril attenuated the effects of CLZ on oxidative stress parameters, NO and serum and cardiac 8-OHdG levels. Captopril significantly attenuated the effect of CLZ on all measured parameters in a dose-dependent manner. These results suggested that captopril exerts a protective action against CLZ-induced myocarditis. Multiple mechanisms contribute to this effect, including a decrease in cardiac oxidative stress and proinflammatory cytokines production, modulation of antioxidant status and protection from oxidative DNA damage. Hence, captopril may be effective in reducing the incidence and severity of CLZ-induced myocarditis in humans.

Oxidatively Generated DNA Lesions as Potential Biomarkers of In Vivo Oxidative Stress

During the last three decades there was an increasing interest for developing biomarkers of oxidative stress. Therefore, efforts have been made to develop sensitive methods aimed at measuring cellular levels of oxidatively generated DNA lesions. Initially, most attention had focused on 8-oxo-7,8-dihydro-2'- deoxyguanosine (8-oxodGuo) probably because reliable analytical methods (mostly HPLC coupled to electrochemical detection) were available since mid-eighties to detect that lesion at the cellular level. With the recent development of more versatile analytical (using mass spectrometric detection) and biochemical assays (such as the comet assay) efforts are currently made to measure simultaneously several DNA lesions. The main degradation pathways of the four main pyrimidine (thymine, cytosine) and purine (adenine, guanine) bases mediated by hydroxyl radical (•OH), one-electron oxidants and singlet oxygen (1O2) have been also studied in detail and results indicate that other DNA modification than 8-oxodGuo could represent suitable biomarkers of oxidative stress. In this review article, the main oxidative degradation products of DNA will be presented together with their mechanisms of formation. Then the developed methods aimed at measuring cellular levels of oxidatively generated DNA lesions will be critically reviewed based on their specificity, versatility and sensitivity. Illustration of the powerfulness of the described methods will be demonstrated using quantification of DNA lesions in cells exposed to ionizing radiations. In addition, recent work highlighting the possible formation of complex DNA lesions will be reported and commented regarding the possibility of using such complex damage as potential biomarkers of oxidative stress.

CRISPR interference directs strand specific spacer acquisition.

BackgroundCRISPR/Cas is a widespread adaptive immune system in prokaryotes. This system integrates short stretches of DNA derived from invading nucleic acids into genomic CRISPR loci, which function as memory of previously encountered invaders. In Escherichia coli, transcripts of these loci are cleaved into small RNAs and utilized by the Cascade complex to bind invader DNA, which is then likely degraded by Cas3 during CRISPR interference.ResultsWe describe how a CRISPR-activated E. coli K12 is cured from a high copy number plasmid under non-selective conditions in a CRISPR-mediated way. Cured clones integrated at least one up to five anti-plasmid spacers in genomic CRISPR loci. New spacers are integrated directly downstream of the leader sequence. The spacers are non-randomly selected to target protospacers with an AAG protospacer adjacent motif, which is located directly upstream of the protospacer. A co-occurrence of PAM deviations and CRISPR repeat mutations was observed, indicating that one nucleotide from the PAM is incorporated as the last nucleotide of the repeat during integration of a new spacer. When multiple spacers were integrated in a single clone, all spacer targeted the same strand of the plasmid, implying that CRISPR interference caused by the first integrated spacer directs subsequent spacer acquisition events in a strand specific manner.ConclusionsThe E. coli Type I-E CRISPR/Cas system provides resistance against bacteriophage infection, but also enables removal of residing plasmids. We established that there is a positive feedback loop between active spacers in a cluster – in our case the first acquired spacer - and spacers acquired thereafter, possibly through the use of specific DNA degradation products of the CRISPR interference machinery by the CRISPR adaptation machinery. This loop enables a rapid expansion of the spacer repertoire against an actively present DNA element that is already targeted, amplifying the CRISPR interference effect.

Identification and Cleavage Site Analysis of DNA Sequences Bound Strongly by Bleomycin

A hairpin DNA library containing an 8-base pair sequence-randomized region was employed in a SELEX-type procedure to identify DNAs that bound strongly to bleomycin A(5), the latter of which was immobilized on a solid support. Ten hairpin DNAs that bound BLM A(5) strongly were identified and sequenced, and used to characterize DNA binding by the antitumor antibiotic. While all 10 selected hairpin DNAs bound to BLM strongly, they did exhibit a range of binding specificities. Further, while the binding specificity was generally the greatest for hairpin DNAs that contained at least one of the sequences (5'-GC-3' and 5'-GT-3') cleaved most frequently by Fe(II).bleomycin, the hairpin DNA exhibiting the poorest binding specificity also contained a 5'-GT-3' site. Four of the hairpin DNA substrates were 5'-(32)P end-labeled and used to assess the preferred sites of cleavage by Fe(II).BLM. The substrate DNAs included two lacking any 5'-GT-3' or 5'-GC-3' site; these were cleaved at 5'-AA-3' and (more strongly) at 5'-AT-3' and 5'-GA-3' sequences. For two hairpin DNAs containing 5'-GT-3' or 5'-GC-3' sequences, cleavage was observed at these sequences as well, but the three aforementioned sequences were also cleaved efficiently. For hairpin DNA 3, which was bound the least well of the 10 DNAs studied, a 5'-TA-3' site was also cleaved efficiently. Thus, the pattern and intensities of cleavage of the four DNAs studied were not entirely consistent with the preferred pattern of DNA cleavage reported for Fe(II).BLM in numerous published studies that have employed arbitrarily chosen DNA substrates. Also studied were the chemistry of DNA cleavage for one of the hairpin DNAs, and competition experiments in which the diminution of cleavage was measured following admixture of a molar excess of a smaller hairpin DNA shown to be an exceptionally good substrate for cleavage by Fe(II).BLM. In the aggregate, the data indicate that the relationship between DNA binding and degradation by Fe.BLM, as well as the chemistry leading to DNA degradation, are more complex than suggested by earlier studies that employed only DNA degradation product analysis as an end point.

The DNA degradation product uric acid directly promotes T cell autoreactivity (90.1)

Abstract Purpose: Abnormally high serum uric acid levels have been associated with several disease conditions including gout and kidney stone disease. More recently, it was shown that uric acid crystals stimulate dendritic cell maturation, activate the NALP3 inflammasome, and enhance antigen-specific immune response. We hypothesize that uric acid can also stimulate T cells directly and in the absence of antigen presentation. Methods: Purified primary human T cells were incubated with and without uric acid at concentrations of 50, 100, 150 and 200 μg/ml. This range of uric acid concentrations covers the normal physiologic serum uric acid concentration as well as a spectrum of hyperuricemic values in human. The expression of T cell activation markers CD25 and CD70 was assessed by flow cytometry. Purified human T cell proliferation response to the various uric acid concentrations was quantified using a colorimetric proliferation assay. Results: Uric acid treatment resulted in antigen-independent T cell activation as determined by increased expression of CD25 in both CD4+ as well as CD8+ T cells. CD4+ T cells also overexpressed CD70, a co-stimulation molecule important in T cell-dependent B cell activation. In addition, we demonstrate a dose response relationship between T cell proliferation and uric acid concentration in vitro. Conclusion: The finding that uric acid directly promotes T cell proliferation and activation in an antigen-independent system is novel and might play a mechanistic role in the inflammatory response observed in gout and other uric acid associated diseases. This work is supported by NIH Grant Number P20-RR015577 and the University of Oklahoma Health Sciences Center.

Degradation of DNA during the autolysis of Saccharomyces cerevisiae.

The autolysis of yeast cells has practical implications in the production of fermented foods and beverages and flavourants for food processing. Protein and RNA degradation during yeast autolysis are well described but the fate of DNA is unclear. Yeast cells ( Saccharomyces cerevisiae) were autolysed by incubating suspensions at 30-60 degrees C (pH 7.0), and at pH 4.0-7.0 (40 degrees C) for 10-14 days. Up to 55% of total DNA was degraded, with consequent leakage into the extracellular environment of mainly 3'- and 5'-deoxyribonucleotides, and lesser amounts of polynucleotides. The rate and extent of DNA degradation, composition of the DNA degradation products and DNase activity were affected by temperature and pH. The highest amount of DNA degradation occurred at 40 degrees C and pH 7.0, where the highest DNase activity was recorded. DNase activity was lowest at 60 degrees C and pH 4.0, where the proportion of polynucleotides in the degradation products was higher.

Synthesis of isotopically labelled DNA degradation products for use in mass spectrometric studies of cellular DNA damage

SUMMARY Thirteen substituted purines and pyrimidines bearing from three to five carbon, nitrogen and/or deuterium isotopic labels have been synthesized in yields ranging from .l to 70%. Most of the products originate from the same small number of commercially available labelled starting materials, and in several cases one intermediate leads to two products, thus minimizing the expense and time required. The parent compounds are found in tissue as the result of DNA damage often linked with carcinogenesis and mutagenesis. The synthesized compounds serve as internal standards for the study of DNA damage using mass spectrom

Detoxication of base propenals and other alpha, beta-unsaturated aldehyde products of radical reactions and lipid peroxidation by human glutathione transferases.

Radiation and chemical reactions that give rise to free radicals cause the formation of highly cytotoxic base propenals, degradation products of DNA. Human glutathione transferases (GSTs; RX:glutathione R-transferase, EC 2.5.1.18) of classes Alpha, Mu, and Pi were shown to promote the conjugation of glutathione with base propenals and related alkenes. GST P1-1 was particularly active in catalyzing the reactions with the propenal derivatives, and adenine propenal was the substrate giving the highest activity. The catalytic efficiency of GST P1-1 with adenine propenal (kcat/Km = 7.7 x 10(5) M-1.s-1) is the highest so far reported with any substrate for this enzyme. In general, GST A1-1 and GST M1-1, in contrast to GST P1-1, were more active with 4-hydroxyalkenals (products of lipid peroxidation) than with base propenals. The adduct resulting from the Michael addition of glutathione to the alkene function of one of the base propenals (adenine propenal) was identified by mass spectrometry. At the cellular level, GST P1-1 was shown to provide protection against alpha, beta-unsaturated aldehydes. GST P1-1 added to the culture medium of HeLa cells augmented the protective effect of glutathione against the toxicity of adenine propenal and thymine propenal. No protective effect of the enzyme was observed in the presence of the competitive inhibitor S-hexylglutathione. GST P1-1 introduced into Hep G2 cells by electroporation was similarly found to increase their resistance to acrolein. The results show that glutathione transferases may play an important role in cellular detoxication of electrophilic alpha, beta-unsaturated carbonyl compounds produced by radical reactions, lipid peroxidation, ionizing radiation, and drug metabolism.

Phage φ92: A New Choleraphage Infecting &lt;i&gt;Vibrio cholerae&lt;/i&gt; Cells Belonging to Serovars O1 and Non-O1

A new choleraphage, psi 92, with an icosahedral head and no tail structure has been described. This phage has a wide host range. It infects and lyses strains belonging to different serovars and biotypes of Vibrio cholerae. The phage utilises host DNA degradation products for its own DNA synthesis. psi 92 contains a linear double-stranded DNA of 56 kbp. A physical map of the phage genome has been constructed using the restriction enzyme KpnI. Intracellular development of phage psi 92 following infection was examined by thin-section electron microscopy.

Introduction of a UV-damaged replicon into a recipient cell is not a sufficient condition to produce an SOS-inducing signal

Three models have been proposed for the nature of the SOS-inducing signal in E. coli. One model postulates that degradation products of damaged DNA generate an SOS-inducing signal; another model surmises that the very lesions produced by UV damage constitute the SOS-inducing signal in vivo; a third model proposes that DNA damage is processed upon DNA replication to form single-stranded DNA (the SOS signal) that activates RecA protein.We tested the models by measuring SOS induction produced by introducing into recipient cells the UV-damaged DNA of 2 constructed phagemids. We used phagemids since they transferred DNA to the recipients with 100% efficiency. The origin of replication of the phagemids was either oriC from the E. coli chromosome, or oriF from F plasmid. Replication of the oriC phagemid was dependent on methylation. A UV-damaged oriC phagemid failed to induce SOS functions in a recipient cell whereas an oriF phagemid did induce them. Our results disprove the first and the second model proposed for the nature of the SOS-inducing signal. The failure of a UV-damaged oriC replicon to induce SOS can be explained by the third model if one assumes that replication of a UV-damaged oriC plasmid does not generate single-stranded DNA as does the E. coli chromosome after UV damage.