Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

Interaction of Multiwalled Carbon Nanotube Produces Structural Alteration of DNA

Multiwalled carbon nanotube (MWCNT) has been found to produce structural changes in Calf Thymus-DNA (CT-DNA). The interaction or binding of the multi-walled carbon nanotubes (MWCNT) was investigated in order to discover if it brings about any significant changes of the DNA double helix using CD spectra of the CT-DNA at two concentration levels of MWCNT representing an increasing MWCNT/DNA molar ratio. In addition, spectrophotometric titrations between MWCNT and CT-DNA were carried out in order to utilize spectral changes as a means of detecting specific binding modes of either intercalation or degradation of DNA. Interactions of MWCNT induced significant changes in the CD spectra of the B-form of natural DNA. The intensities of the positive CD band at 280 nm decreased significantly. This decrease was found to be concentration-dependent. Following spectrophotometric titrations; specific subtle conformational changes were observed with a molar ratio combination of 2:1 between MWCNT and CT-DNA and these were characterized by a formation constant of the order of 103 M-1 and a negative Gibbs free energy suggesting that MWCNT avidly binds to DNA. Thermodynamic considerations revealed that electrostatic interactions between the DNA base pairs and the MWCNT are taking place accounting for the negative free energy change, positive enthalpy change with a small entropy change. The results obtained in the study of the binding interactions of MWCNT with DNA confirm that a cytogenetic effect of MWCNT with DNA is a possibility in vivo.

Vibration-induced extra torque during electrically-evoked contractions of the human calf muscles

Background: High-frequency trains of electrical stimulation applied over the lower limb muscles can generate forces higher than would be expected from a peripheral mechanism (i.e. by direct activation of motor axons). This phenomenon is presumably originated within the central nervous system by synaptic input from Ia afferents to motoneurons and is consistent with the development of plateau potentials. The first objective of this work was to investigate if vibration (sinusoidal or random) applied to the Achilles tendon is also able to generate large magnitude extra torques in the triceps surae muscle group. The second objective was to verify if the extra torques that were found were accompanied by increases in motoneuron excitability. Methods: Subjects (n = 6) were seated on a chair and the right foot was strapped to a pedal attached to a torque meter. The isometric ankle torque was measured in response to different patterns of coupled electrical (20-Hz, rectangular 1-ms pulses) and mechanical stimuli (either 100-Hz sinusoid or gaussian white noise) applied to the triceps surae muscle group. In an additional investigation, M(max) and F-waves were elicited at different times before or after the vibratory stimulation. Results: The vibratory bursts could generate substantial self-sustained extra torques, either with or without the background 20-Hz electrical stimulation applied simultaneously with the vibration. The extra torque generation was accompanied by increased motoneuron excitability, since an increase in the peak-to-peak amplitude of soleus F waves was observed. The delivery of electrical stimulation following the vibration was essential to keep the maintained extra torques and increased F-waves. Conclusions: These results show that vibratory stimuli applied with a background electrical stimulation generate considerable force levels (up to about 50% MVC) due to the spinal recruitment of motoneurons. The association of vibration and electrical stimulation could be beneficial for many therapeutic interventions and vibration-based exercise programs. The command for the vibration-induced extra torques presumably activates spinal motoneurons following the size principle, which is a desirable feature for stimulation paradigms.

Cow/calf preweaning efficiency of Nellore and Bos taurus x Bos indicus crosses

The objectives of this study were to determine if percentage Bos taurus (0 or 50%) of the cow had an effect on ME requirements and milk production, and to compare cow/calf efficiency among 3 mating systems. Metabolizable energy requirements were estimated during a feeding trial that encompassed a gestation and lactation feeding trial for each of 2 groups of cows. Cows were 0 or 50% Bos taurus ( 100 or 50% Nellore) breed type: Nellore cows (NL; n = 10) mated to Nellore bulls, NL cows ( n = 9) mated to Angus bulls, Angus x Nellore (ANL; n = 10) and Simmental x Nellore (SNL; n = 10) cows mated to Canchim (5/ 8 Charolais 3/ 8 Zebu) bulls. Cows were individually fed a total mixed diet that contained 11.3% CP and 2.23 Mcal of ME/kg of DM. At 14-d intervals, cows and calves were weighed and the amount of DM was adjusted to keep shrunk BW and BCS of cows constant. Beginning at 38 d of age, corn silage was available to calves ad libitum. Milk production at 42, 98, 126, and 180 d postpartum was measured using the weigh-suckle-weigh technique. At 190 d of age, calves were slaughtered and body composition estimated using 9-10-11th-rib section to obtain energy deposition. Regression of BW change on daily ME intake (MEI) was used to estimate MEI at zero BW change. Increase in percentage Bos taurus had a significant effect on daily ME requirements (Mcal/d) during pregnancy (P < 0.01) and lactation (P < 0.01). Percentage Bos taurus had a positive linear effect on maintenance requirements of pregnant (P = 0.07) and lactating (P < 0.01) cows; during pregnancy, the ME requirements were 91 and 86% of those in lactation (131 +/- 3.5 vs. 145 +/- 3.4 Mcal.kg(-0.75).d(-1)) for the 0 and 50% B. taurus groups, respectively. The 50% B. taurus cows, ANL and SNL, suckling crossbred calves had greater total MEI (4,319 +/- 61 Mcal; P < 0.01) than 0% B. taurus cows suckling NL (3,484 +/- 86 Mcal) or ANL calves (3,600 +/- 91 Mcal). The 0% B. taurus cows suckling ANL calves were more efficient (45.3 +/- 1.6 g/Mcal; P = 0.03) than straightbred NL (35.1 +/- 1.5 g/Mcal) and ANL or SNL pairs (41.0 +/- 1.0 g/Mcal). Under the conditions of this study, crossbreeding improved cow/ calf efficiency and showed an advantage for cows that have lower energy requirements.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties.

Essential Oil of Thymus vulgaris: Preparation of Pharmaceutical Mouthwash Formulation and In Vitro Evaluation of the Bacterial Plaque-Inhibiting Properties. The aim of this study was to evaluate the in vitro effect of the essential oil of Thymus vulgaris (thyme) pure or incorporate in a alcohol-free pharmaceutical mouthwash formulation, against Streptococcus mutans (ATCC 25175), being determined the Minimal Inhibitory Concentration (MIC) and the effect in the bacterial plate formation. The MIC value obtained for the essential oil was 100 mu g/mL (1 %). The mouthwash pharmaceutical formulation containing commercial essential oil of T. vulgaris was preparated. Microbiological and macroscopic analysis as well as analyses for MEV confirmed the effectiveness of this new alcohol-free mouthwash formulation containing essential oil of T. vulgaris as agent with plaque-inhibiting properties and possible application in the preventive dentistry. The chemical characterization of the bioactive essential oil was accomplished by CG-MS, being verified the presence of carvacrol, p-cimene and alpha-pinene as major constituents.

Structure and distribution of N-glycans on the S-7-allele stylar self-incompatibility ribonuclease of Nicotiana alata

S-RNases are the stylar products of the self-incompatibility (S)-locus in solanaceous plants (including Nicotiana alata), and as such, are involved in the prevention of self-pollination. All cDNA sequences of S-RNase products of functional S-alleles contain potential N-glycosylation sites, with one site being conserved in all cases, suggesting that N-glycosylation is important in self-incompatibility. In this study, we report on the structure and localization of the N-glycans on the S-7-allele RNase of N, alata, A total of nine N-glycans, belonging to the high-mannose- and xylosylated hybrid-classes, were identified and characterized by a combination of electrospray-ionization mass-spectrometry (ESI-MS), H-1-NMR spectroscopy, and methylation analyses. The glycosylation pattern of individual glycosylation sites was determined by ESI-MS of the glycans released from isolated chymotryptic glycopeptides, All three N-glycosylation sites showed microheterogeneity and each had a unique complement of N-glycans, The N-glycosylation pattern of the S-7-RNase is significantly different to those of the S-1- and S-2-RNases.

Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited

Decreased recent thymus emigrant number in rheumatoid factor-negative polyarticular juvenile idiopathic arthritis

Objectives To determine TCR excision circle (TREC) levels, a marker of recent thymic emigrants, in the peripheral lymphocyte pool of rheumatoid factor-negative (RF circle divide) polyarticular juvenile idiopathic arthritis (JIA) children. Materials and methods We studied TREC levels in peripheral blood mononuclear cells (PBMC) in 30 RF circle divide polyarticular JIA children with active disease and in 30 age- and gender-matched healthy controls. Signal-joint TREC concentration was determined by real-time quantitative-PCR as the number of TREC copies/mu g PBMC DNA gauged by a standard curve with known number of TREC-containing plasmids. Results TREC levels in PBMC were significantly lower in JIA (4.90 +/- 3.86 x 10(4) TRECs/mu g DNA) as compared to controls (10.45 +/- 8.45 x 10(4) TRECs/mu g DNA, p=0.001). There was an inverse correlation between age and TREC levels in healthy children (r=-0.438, p=0.016) but not in JIA. No clinical association was observed between TREC levels and disease activity and use of oral steroids and methotrexate. Conclusions The finding of decreased PBMC TREC levels in RF circle divide polyarticular JIA children is consistent with a low proportion of recent thymus emigrants. This may interfere with the equilibrium between populations of polyclonal and naive T cells versus oligoclonal memory auto-reactive T cells and, therefore, may hinder the maintenance of immune tolerance in this disease.

Immunoendocrinology of the Thymus in Chagas Disease

During immune response to infectious agents, the host develops an inflammatory response which could fail to eliminate the pathogen or may become dysregulated. In this case, the ongoing response acquires a new status and turns out to be detrimental. The same elements taking part in the establishment and regulation of the inflammatory response (cytokines, chemokines, regulatory T cells and counteracting compounds like glucocorticoids) may also mediate harmful effects. Thymic disturbances seen during Trypanosoma cruzi (T. cruzi) infection fit well with this conceptual framework. After infection, this organ suffers a severe atrophy due to apoptosis-induced thymocyte exhaustion, mainly affecting the immature double-positive (DP) CD4+CD8+ population. Thymus cellularity depletion, which occurs in the absence of main immunological mediators involved in anti-T. cruzi defense, seems to be linked to a systemic cytokine/hormonal imbalance, involving a dysregulated increase in Tumor Necrosis Factor alpha (TNF-alpha) and corticosterone hormone levels. Additionally, we have found an anomalous exit of potentially autoimmune DP cells to the periphery, in parallel to a shrinkage in the compartment of natural regulatory T cells. In this context, our data clearly point to the view that the thymus is a target organ of T. cruzi infection. Preserved thymus may be essential for the development of an effective immune response against T. cruzi, but this organ is severely affected by a dysregulated circuit of proinflammatory cytokines and glucocorticoids. Also, the alterations observed in the DP population might have potential implications for the autoimmune component of human Chagas disease. Copyright (C) 2011 S. Karger AG, Basel

Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.

13,14-Dihydro-15-Keto Prostaglandin F(2)alpha Release in Response to Oxytocin Challenge Early Post-Partum in Anoestrous Nelore Cows Submitted to Temporary Calf Removal and Progesterone Priming

The study evaluated, in early post-partum anoestrous Nelore cows, if the increase in plasma oestradiol (E2) concentrations in the pre-ovulatory period and/or progesterone priming (P4 priming) preceding ovulation, induced by hormonal treatment, reduces the endogenous release of prostaglandin PGF(2)alpha and prevents premature lysis of the corpus luteum (CL). Nelore cows were subjected to temporary calf removal for 48 h and divided into two groups: GPE/eCG group (n = 10) and GPG/eCG group (n = 10). Animals of the GPE/eCG group were treated with a GnRH agonist. Seven days later, they received 400 ID of eCG, immediately after PGF(2)alpha treatment, and on day 0, 1.0 mg of oestradiol benzoate (EB). Cows of the GPG/eCG group were similarly treated as those of the GPE/eCG group, except that EB was replaced with a second dose of GnRH. All animals were challenged with oxytocin (OT) 9, 12, 15 and 18 days after EB or GnRH administration and blood samples were collected before and 30 min after OT. Irrespective of the treatments, a decline in P4 concentration on day 18 was observed for cows without P4 priming. However, animals exposed to P4 priming, treated with EB maintained high P4 concentrations (8.8 +/- 1.2 ng/ml), whereas there was a decline in P4 on day 18 (2.1 +/- 1.0 ng/ml) for cows that received GnRH to induce ovulation (p < 0.01). Production of 13,14-dihydro-15-keto prostaglandin F(2)alpha (PGFM) in response to OT increased between days 9 and 18 (p < 0.01), and this increase tended to be more evident in animals not exposed to P4 priming (p < 0.06). In conclusion, the increase in E2 during the pre-ovulatory period was not effective in inhibiting PGFM release, which was lower in P4-primed than in non-primed animals. Treatment with EB promoted the maintenance of elevated P4 concentrations 18 days after ovulation in P4-primed animals, indicating a possible beneficial effect of hormone protocols containing EB in animals with P4 priming.

Composition and antioxidant activity of thymus vulgaris volatiles: comparison between supercritical fluid extraction and hydrodistillation

Supercritical fluid extraction (SEE) of the volatile oil from Thymus vulgaris L. aerial flowering parts was performed under different conditions of pressure, temperature, mean particle size and CO2 flow rate and the correspondent yield and composition were compared with those of the essential oil isolated by hydrodistillation (HD). Both the oils were analyzed by GC and GC-MS and 52 components were identified. The main volatile components obtained were p-cymene (10.0-42.6% for SFE and 28.9-34.8% for HD), gamma-terpinene (0.8-6.9% for SFE and 5.1-7.0% for HD), linalool (2.3-5.3% for SFE and 2.8-3.1% for HD), thymol (19.5-40.8% for SFE and 35.4-41.6% for HD), and carvacrol (1.4-3.1% for SFE and 2.6-3.1% for HD). The main difference was found to be the relative percentage of thymoquinone (not found in the essential oil) and carvacryl methyl ether (1.0-1.2% for HD versus t-0.4 for SFE) which can explain the higher antioxidant activity, assessed by Rancimat test, of the SFE volatiles when compared with HD. Thymoquinone is considered a strong antioxidant compound.