A dynamic checkpoint in oxidative lesion discrimination by formamidopyrimidine-DNA glycosylase.

In contrast to proteins recognizing small-molecule ligands, DNA-dependent enzymes cannot rely solely on interactions in the substrate-binding centre to achieve their exquisite specificity. It is widely believed that substrate recognition by such enzymes involves a series of conformational changes in the enzyme-DNA complex with sequential gates favoring cognate DNA and rejecting nonsubstrates. However, direct evidence for such mechanism is limited to a few systems. We report that discrimination between the oxidative DNA lesion, 8-oxoguanine (oxoG) and its normal counterpart, guanine, by the repair enzyme, formamidopyrimidine-DNA glycosylase (Fpg), likely involves multiple gates. Fpg uses an aromatic wedge to open the Watson-Crick base pair and everts the lesion into its active site. We used molecular dynamics simulations to explore the eversion free energy landscapes of oxoG and G by Fpg, focusing on structural and energetic details of oxoG recognition. The resulting energy profiles, supported by biochemical analysis of site-directed mutants disturbing the interactions along the proposed path, show that Fpg selectively facilitates eversion of oxoG by stabilizing several intermediate states, helping the rapidly sliding enzyme avoid full extrusion of every encountered base for interrogation. Lesion recognition through multiple gating intermediates may be a common theme in DNA repair enzymes.

Active destabilization of base pairs by a DNA glycosylase wedge initiates damage recognition.

Formamidopyrimidine-DNA glycosylase (Fpg) excises 8-oxoguanine (oxoG) from DNA but ignores normal guanine. We combined molecular dynamics simulation and stopped-flow kinetics with fluorescence detection to track the events in the recognition of oxoG by Fpg and its mutants with a key phenylalanine residue, which intercalates next to the damaged base, changed to either alanine (F110A) or fluorescent reporter tryptophan (F110W). Guanine was sampled by Fpg, as evident from the F110W stopped-flow traces, but less extensively than oxoG. The wedgeless F110A enzyme could bend DNA but failed to proceed further in oxoG recognition. Modeling of the base eversion with energy decomposition suggested that the wedge destabilizes the intrahelical base primarily through buckling both surrounding base pairs. Replacement of oxoG with abasic (AP) site rescued the activity, and calculations suggested that wedge insertion is not required for AP site destabilization and eversion. Our results suggest that Fpg, and possibly other DNA glycosylases, convert part of the binding energy into active destabilization of their substrates, using the energy differences between normal and damaged bases for fast substrate discrimination.

Cat Scratch Disease in kidney transplant receptors: is it a rare or underdiagnosed pathology?

Cat Scratch Disease (CSD) is an infectious disorder which appears after cat scratching particularly in children and adolescents. Bartonella henselae is the etiologic agent more frequently involved. There are only a few recent reports demonstrating the disease after transplantation, although the illness is not infrequent in immunologically competent people. Indeed CSD in transplant receptors has only been recently emphasized in the literature and it was concluded that fever and lymphadenopathy in patients who had been exposed to cats should prompt clinicians to maintain a suspicion for the infection. In this report CSD infecting a renal transplanted adolescent complaining of headache, blurred vision and fever, presenting a cat scratching lesion in the right arm, with a bilateral painful cervical lymphadenopathy was related. He also presented indirect immunofluorescency identifying that the two subtype's titles of Bartonella--henselae and quintana--were elevated. Treatment with doxicicline e rifampicin was introduced and the patient became asymptomatic in about 3 weeks.

Cat Scratch Disease in kidney transplant receptors: is it a rare or underdiagnosed pathology? / Doença da Arranhadura do Gato em transplantados de rim: patologia rara ou subdiagnosticada?

Cat Scratch Disease (CSD) is an infectious disorder which appears after cat scratching particularly in children and adolescents. Bartonella henselae is the etiologic agent more frequently involved. There are only a few recent reports demonstrating the disease after transplantation, although the illness is not infrequent in immunologically competent people. Indeed CSD in transplant receptors has only been recently emphasized in the literature and it was concluded that fever and lymphadenopathy in patients who had been exposed to cats should prompt clinicians to maintain a suspicion for the infection. In this report CSD infecting a renal transplanted adolescent complaining of headache, blurred vision and fever, presenting a cat scratching lesion in the right arm, with a bilateral painful cervical lymphadenopathy was related. He also presented indirect immunofluorescency identifying that the two subtype's titles of Bartonella-henselae and quintana- were elevated. Treatment with doxicicline e rifampicin was introduced and the patient became asymptomatic in about 3 weeks.

A Doença da Arranhadura do Gato (DAG) é uma desordem infecciosa que surge após a arranhadela do animal, especialmente em crianças e adolescentes. Bartonella hanselae é o agente etiológico mais frequentemente envolvido. Há somente poucos relatos recentes demonstrando a doença após transplante renal, embora a mesma não seja infrequente em pessoas imunologicamente competentes. Na verdade, DAG em receptores de transplantes tem sido somente recentemente enfatizada na literatura e concluiu-se que a presença de febre e linfadenopatias em pacientes que estiveram expostos a contato com o animal deveriam sinalizar os clínicos a manter suspeita da infecção. Neste relato de DAG, é exposto o caso de um adolescente transplantado de rim queixando-se de cefaleia, visão borrosa e febre, apresentando cicatriz de arranhadura no braço direito, com linfadenopatia cervical bilateral dolorosa. Também apresentava imunoflorescência indireta identificando que os dois subtipos de Bartonella- a henselae e a quintana- estavam elevadas. Tratamento com doxiciclina e rifampicina foi iniciado e o paciente tornou-se assintomático em cerca de três semanas.

Ramsay Hunt syndrome with facial vesicular rash: a unique clinical presentation in a kidney transplant patient.

Ramsay Hunt Syndrome (RHS) is the result of herpes zoster virus reactivation producing hearing loss, pain and vesicles in the ear or mouth, along with ipsilateral facial palsy due to the 7th cranial nerve geniculate ganglion infectious involvement. This condition has not been previously described, particularly in transplant patients. A 38-year old man underwent kidney transplantation and two years later experienced an ache on the left side of the face and hearing loss in the ear, also exhibiting vesicular lesions and concomitant facial peripheral palsy. Acyclovir IV was initiated, and the prednisone dose was increased. The patient was discharged 15 days later, feeling better but still exhibiting dark spots on his face. At three months follow-up he was asymptomatic, showing notable palsy improvement. Until this case, herpes zoster facial lesions causing typical RHS have never been reported in literature, particularly in kidney transplant patients.

Structure and stability of duplex DNA containing (5'S)-5',8-cyclo-2'-deoxyadenosine: an oxidatively generated lesion repaired by NER.

Cellular respiration and ionizing radiation generate 5',8-cyclo-2'-deoxyribonucleosides, a special type of DNA damage that involves two modifications in the same nucleotide. These lesions evade the action of base excision glycosylases, and their removal is a function of the nucleotide excision repair pathway. Diastereomeric 5',8-cyclo-2'-deoxyadenosine blocks mammalian DNA replication, diminishes the levels of DNA transcription, and induces transcriptional mutagenesis. Using solution state NMR spectroscopy and restrained molecular dynamics simulations, we have determined the structure of an undecameric DNA duplex having a centrally located (5'S)-5',8-cyclo-2'-deoxyadenosine residue paired to T. The damaged duplex structure is a right-handed helix having Watson-Crick base-pair alignments throughout, and 2-deoxyribose puckers within the B-form conformation. Only small structural perturbations are observed at the lesion-containing and 5'-flanking base pair. The 2-deoxyribose of the damaged nucleotide adopts the O4'-exo conformation, and the S-cdA·T base pair is propeller twisted. The 5'-lesion-flanking base is tilted forming a significantly buckled base pair with its partner guanine. Analysis of UV-melting curves indicates mild thermal and thermodynamic destabilization on the damaged duplex. The S-cdA·T duplex structure shows many similarities to and some intriguing differences from the recently reported structure of an S-cdG·dC duplex³¹ that suggest different lesion site dynamics.

Solution structure of duplex DNA containing a ß-carba-Fapy-dG lesion.

The addition of hydroxyl radicals to the C8 position of guanine can lead to the formation of a 2,6-diamino-4-hydroxy-5-formamido-2'-deoxypyrimidine (Fapy-dG) lesion, whose endogenous levels in cellular DNA rival those of 8-oxo-7,8-dihydroxy-2'-deoxyguanosine. Despite its prevalence, the structure of duplex DNA containing Fapy-dG is unknown. We have prepared an undecameric duplex containing a centrally located ß-cFapy-dG residue paired to dC and determined its solution structure by high-resolution NMR spectroscopy and restrained molecular dynamic simulations. The damaged duplex adopts a right-handed helical structure with all residues in an anti conformation, forming Watson-Crick base pair alignments, and 2-deoxyribose conformations in the C2'-endo/C1'-exo range. The formamido group of Fapy rotates out of the pyrimidine plane and is present in the Z and E configurations that equilibrate with an approximate 2:1 population ratio. The two isomeric duplexes show similar lesion-induced deviations from a canonical B-from DNA conformation that are minor and limited to the central three-base-pair segment of the duplex, affecting the stacking interactions with the 5-lesion-neighboring residue. We discuss the implications of our observations for translesion synthesis during DNA replication and the recognition of Fapy-dG by DNA glycosylases.

Structure and stability of DNA containing an aristolactam II-dA lesion: implications for the NER recognition of bulky adducts.

Aristolochic acids I and II are prevalent plant toxicants found in the Aristolochiaceae plant family. Metabolic activation of the aristolochic acids leads to the formation of a cyclic N-hydroxylactam product that can react with the peripheral amino group of purine bases generating bulky DNA adducts. These lesions are mutagenic and established human carcinogens. Interestingly, although AL-dG adducts progressively disappear from the DNA of laboratory animals, AL-dA lesions has lasting persistence in the genome. We describe here NMR structural studies of an undecameric duplex damaged at its center by the presence of an ALII-dA adduct. Our data establish a locally perturbed double helical structure that accommodates the bulky adduct by displacing the counter residue into the major groove and stacking the ALII moiety between flanking bases. The presence of the ALII-dA perturbs the conformation of the 5'-side flanking base pair, but all other pairs of the duplex adopt standard conformations. Thermodynamic studies reveal that the lesion slightly decreases the energy of duplex formation in a sequence-dependent manner. We discuss our results in terms of its implications for the repair of ALII-dA adducts in mammalian cells.

Incorporation of 3-aminobenzanthrone into 2'-deoxyoligonucleotides and its impact on duplex stability.

3-Nitrobenzanthrone (3NBA), an environmental pollutant and potent mutagen, causes DNA damage via the reaction of its metabolically activated form with the exocyclic amino groups of purines and the C-8 position of guanine. The present work describes a synthetic approach to the preparation of oligomeric 2'-deoxyribonucleotides containing a 2-(2'-deoxyguanosin-N(2)-yl)-3-aminobenzanthrone moiety, one of the major DNA adducts found in tissues of living organisms exposed to 3NBA. The NMR spectra indicate that the damaged oligodeoxyribonucleotide is capable of forming a regular double helical structure with the polyaromatic moiety assuming a single conformation at room temperature; the spectra suggest that the 3ABA moiety resides in the duplex minor groove pointing toward the 5'-end of the modified strand. Thermodynamic studies show that the dG(N(2))-3ABA lesion has a stabilizing effect on the damaged duplex, a fact that correlates well with the long persistence of this damage in living organisms.

Energetic preference of 8-oxoG eversion pathways in a DNA glycosylase.

Base eversion is a fundamental process in the biochemistry of nucleic acids, allowing proteins engaged in DNA repair and epigenetic modifications to access target bases in DNA. Crystal structures reveal end points of these processes, but not the pathways involved in the dynamic process of base recognition. To elucidate the pathway taken by 8-oxoguanine during base excision repair by Fpg, we calculated free energy surfaces during eversion of the damaged base through the major and minor grooves. The minor groove pathway and free energy barrier (6-7 kcal/mol) are consistent with previously reported results (Qi, Y.; Spong, M. C.; Nam, K.; Banerjee, A.; Jiralerspong, S.; Karplus, M.; Verdine, G. L. Nature 2009, 462, 762.) However, eversion of 8-oxoG through the major groove encounters a significantly lower barrier (3-4 kcal/mol) more consistent with experimentally determined rates of enzymatic sliding during lesion search (Blainey, P. C.; van Oijent, A. M.; Banerjee, A.; Verdine, G. L.; Xie, X. S. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 5752.). Major groove eversion has been suggested for other glycosylases, suggesting that in addition to function, dynamics of base eversion may also be conserved.

Low response to intradermal hepatitis B vaccination in incident hemodialysis patients.

INTRODUCTION: Hepatitis B (HB) may progress to cirrhosis and liver carcinoma. Its prevalence is estimated at 3.2 % in hemodialysis (HD) patients. HB vaccine when applied intramuscularly (IM) in end-stage renal disease patients often does not induce appropriate antibody titers. However, there has been suggestion for intradermal (ID) to be a more effective inoculation method. OBJECTIVE: To compare the immune response to IM or ID vaccine administration on HD patients. PATIENTS AND METHODS: Thirty one incident HD patients were randomly assigned alternately to IM or ID vaccine inoculation. Vaccine doses were applied at three monthly intervals, with patients being followed-up for six months. Sixteen patients were assigned to IM (40 mg/dose) and 15 to ID (4 mg/dose) vaccine administration. HB-virus surface antibody titer, hematimetric parameters, serum urea level and Kt/V were monthly evaluated. C-reactive protein, parathormone, ferritin, aminotransferases and albumin serum levels were evaluated before and at the sixth month of the initial inoculation. RESULTS: Urea levels were significantly higher in the ID group (P(1) = 0.031); ferritin levels were higher in the IM (P(2) = 0.037) and C-reactive protein levels tended to be higher in the ID group. An interim evaluation by the Safety Monitoring Committee recommended discontinuing the study as IM vaccination had converted 62.5% of the exposed subjects, while ID inoculation converted only 13.3%. CONCLUSION: As performed, ID applied vaccine was inferior to the IM inoculation. Such result may depend on the inoculated doses or some other factor, such as inflammation.

Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties.

Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N(6)-(2-Deoxy-α,ß-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and ß-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis.

Baixa resposta da vacinação intradérmica contra hepatite B em pacientes incidentes em hemodiálise / Low response to intradermal hepatitis B vaccination in incident hemodialysis patients

INTRODUÇÃO: A hepatite B pode evoluir para cirrose e hepatocarcinoma. Sua prevalência estimada é de 3,2 por cento em pacientes em hemodiálise (HD). A vacina para hepatite B (HB), quando aplicada por via intramuscular (IM) em pacientes com insuficiência renal crônica fase V, frequentemente não induz produção adequada de anticorpos. A injeção intradérmica (ID) foi sugerida como sendo o método de inoculação mais eficiente. OBJETIVO: Comparar a resposta imune à injeção IM ou ID da vacina em indivíduos em HD. PACIENTES E MÉTODOS: Trinta e um pacientes incidentes em HD foram randomizados alternativamente para vacinação contra HB via IM ou ID. Dezesseis foram designados aleatoriamente para receber vacina IM (40 mg/dose) e 15 ID (4mg /dose). Os níveis de anticorpos de superfície do vírus da hepatite B, parâmetros hematimétricos, ureia sérica, e Kt/V foram avaliados mensalmente. Proteína-C reativa, paratormônio, ferritina, aminotransferases e albumina foram avaliados antes da inoculação inicial e seis meses após a mesma. RESULTADOS: Os níveis de uréia foram maiores no grupo ID (P(1) = 0,031); os níveis de ferritina foram mais elevados no IM (P(2) = 0,037). Houve tendência a aumento nos níveis de proteína C reativa no grupo ID. A avaliação do Comitê de Monitoramento de Segurança dos indivíduos expostos recomendou a suspensão do estudo já que a inoculação por via IM converteu 62,5 por cento e a ID converteu apenas 13,3 por cento dos pacientes expostos. CONCLUSÃO: Com a metodologia utilizada, os resultados da vacina contra HB aplicada por via ID foi inferior à inoculação IM. Tais resultados podem ser decorrentes das doses inoculadas ou de outros fatores, como inflamação.

INTRODUCTION: Hepatitis B (HB) may progress to cirrhosis and liver carcinoma. Its prevalence is estimated at 3.2 percent in hemodialysis (HD) patients. HB vaccine when applied intramuscularly (IM) in end-stage renal disease patients often does not induce appropriate antibody titers. However, there has been suggestion for intradermal (ID) to be a more effective inoculation method. OBJECTIVE: To compare the immune response to IM or ID vaccine administration on HD patients. PATIENTS AND METHODS: Thirty one incident HD patients were randomly assigned alternately to IM or ID vaccine inoculation. Vaccine doses were applied at three monthly intervals, with patients being followed-up for six months. Sixteen patients were assigned to IM (40 mg/dose) and 15 to ID (4 mg/dose) vaccine administration. HB-virus surface antibody titer, hematimetric parameters, serum urea level and Kt/V were monthly evaluated. C-reactive protein, parathormone, ferritin, aminotransferases and albumin serum levels were evaluated before and at the sixth month of the initial inoculation. RESULTS: Urea levels were significantly higher in the ID group (P(1) = 0.031); ferritin levels were higher in the IM (P(2) = 0.037) and C-reactive protein levels tended to be higher in the ID group. An interim evaluation by the Safety Monitoring Committee recommended discontinuing the study as IM vaccination had converted 62.5 percent of the exposed subjects, while ID inoculation converted only 13.3 percent. CONCLUSION: As performed, ID applied vaccine was inferior to the IM inoculation. Such result may depend on the inoculated doses or some other factor, such as inflammation.

Identification of a reduction product of aristolochic acid: implications for the metabolic activation of carcinogenic aristolochic acid.

Aristolochic acids are nephrotoxic and carcinogenic natural products that have been implicated both in endemic nephropathy in the Balkan region and in ailments caused by ingestion of herbal remedies. Aristolochic acids are metabolized to active intermediates that bind to DNA. In this study, reduction of aristolochic acid I with zinc in acetic acid afforded a new product that was characterized as 9-methoxy-7-methyl-2H-1,3-oxazolo[5',4'-10,9]phenanthro[3,4-d]-1,3-dioxolane-5-carboxylic acid, designated as aristoxazole, along with the expected aristolactam I. This new compound is a condensation product of aristolochic acid and acetic acid that may be related to the aristolochic acid-DNA adducts. The proposed mechanism of formation of aristoxazole involves nucleophilic attack of acetic acid on the nitrenium ion of aristolochic acid I. On the basis of these studies, a route to the metabolic activation of aristolochic acids and formation of adducts with DNA in in vitro systems is proposed and discussed.

NMR solution structures of clustered abasic site lesions in DNA: structural differences between 3'-staggered (-3) and 5'-staggered (+3) bistranded lesions.

Ionizing radiation produces a distinctive pattern of bistranded clustered lesions in DNA. A relatively low number of clustered lesions may be lethal to cells when compared to a larger number of single lesions. Enzyme cleavage experiments suggest that the orientation of bistranded lesions causes differential recognition and removal of these lesions. Like that of a previous study of bistranded abasic site lesion [Hazel, R. D., Tian, K., and de los Santos, C. (2008) Biochemistry 47, 11909-11919], the aim of this investigation was to determine the structures of two DNA duplexes each containing two synthetic apurinic/apyrimidinic (AP) residues, positioned on opposite strands and separated by two base pairs. In the first duplex, the AP residues are staggered in the 3' orientation [-3 duplex, (AP)(2)-3 duplex], while in the second duplex, the AP residues are staggered in the 5' orientation [+3 duplex, (AP)(2)+3 duplex]. NOESY spectra recorded in 100 and 10% D(2)O buffer solutions allowed the assignment of the nonexchangeable and exchangeable protons, respectively, for each duplex. Cross-peak connectivity in the nonexchangeable proton spectra indicates that the duplex is a regular right-handed helix with the AP residues and orphan bases located inside the duplexes. The exchangeable proton spectra establish the formation of Watson-Crick G·C alignment for the two base pairs between the lesion sites in both duplexes. Distance-restrained molecular dynamics simulation confirmed the intrahelical orientations of the AP residues. The proximity of the AP residues across the minor groove of the -3 duplex and across the major groove in the +3 duplex is similar to their locations in the case of -1 and +1 clusters. This difference in structure may be a key factor in the differential recognition of bistranded AP lesions by human AP endonuclease.

Streptococcus agalactiae: a rare peritoneal infection in a continuous ambulatory peritoneal dialysis patient.

Streptococcus agalactiae causes a rare and often fatal peritonitis in continuous ambulatory peritoneal dialysis (CAPD). A 52-year-old white female with Alport and chronic kidney disease was initiated on CAPD treatment. Nineteen months later she had a S. agalactiae peritonitis identified and received initially gentamicin-cephalothin, which was changed to ceftazidime, tobramycin, and vancomycin. Recovery started after peritoneal catheter removal. After 3 weeks, severe leucopenia occurred. Granulokine and steroids were given. Six weeks later, she felt well and an abdominal video laparoscopic procedure disclosed a diffuse peritoneal fibrosis, precluding CAPD resumption. She is now doing well on hemodialysis (HD).

Stereoselective nucleoside deuteration for NMR studies of DNA.

A procedure has been elaborated for stereoselective deuterium substitution of one of the diastereotopic 5'-protons in 2'-deoxynucleotides. The synthetic scheme uses the reduction of the 5-oxosugar derivative with deuterated Alpine-Borane. The resulting deuterosugar is converted into pyrimidine nucleosides and incorporated into DNA using standard protocols. Comparison of two-dimensional NMR spectra of the fully protonated and partially deuterated duplexes allowed us to assign diastereotopic 5' protons, increasing the number of experimental restraints used for structure determination.

NMR structure of duplex DNA containing the alpha-OH-PdG.dA base pair: a mutagenic intermediate of acrolein.

Acrolein, a cell metabolic product and main component of cigarette smoke, reacts with DNA generating alpha-OH-PdG lesions, which have the ability to pair with dATP during replication thereby causing G to T transversions. We describe the solution structure of an 11-mer DNA duplex containing the mutagenic alpha-OH-PdG.dA base pair intermediate, as determined by solution nuclear magnetic resonance (NMR) spectroscopy and retrained molecular dynamics (MD) simulations. The NMR data support a mostly regular right-handed helix that is only perturbed at its center by the presence of the lesion. Undamaged residues of the duplex are in anti orientation, forming standard Watson-Crick base pairs alignments. Duplication of proton signals at and near the damaged base pair reveals the presence of two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. The alpha-OH-PdG adduct assumes a syn conformation pairing to its partner dA base that is protonated at pH 6.6. The three-dimensional structure obtained by restrained molecular dynamics simulations show hydrogen bond interactions that stabilize alpha-OH-PdG in a syn conformation and across the lesion containing base pair. We discuss the implications of the structures for the mutagenic bypass of acrolein lesions.

Cardiovascular risk factors and carotid intima-media thickness in asymptomatic children.

Atherosclerosis, beginning in childhood, is dependent on several risk factors and may be predictive of coronary artery disease in adulthood. The risk factors for subclinical atherosclerosis are similar to those for clinical disease. Carotid intima-media thickness is a measure of subclinical atherosclerosis and a predictor of subsequent vascular events. This study aimed to examine the relationships of carotid intima-media thickness with known risk factors in asymptomatic children. Family history of cardiovascular disease was collected, together with anthropometric, demographic, and clinical data. Body mass index z-scores were calculated. Serum glucose, lipid fractions, fibrinogen, and C-reactive protein were determined. High-resolution ultrasonography was used to assess intima-media thickness. Associations and relationships of risk factors with composite intima-media thickness were explored. The study enrolled 93 children (44 girls) ranging in age from 49 to 169 months. The boys had a thicker intima-media (0.46 +/- 0.06 mm) than the girls (0.43 +/- 0.06 mm; p = 0.028). The unadjusted triglyceride levels were significantly higher in the overweight and obese children (p = 0.010). Body mass index and overweight/obesity were positively related to intima-media thickness (r = 0.259; p = 0.012 and r (s) = 0.230; p = 0.027, respectively), whereas family history of cardiovascular disease was unrelated. Only gender and overweight/obesity were related to intima-media thickness in a multiple linear regression model (R (2) = 0.125; p = 0.002). Male gender and overweight/obesity were associated with increased intima-media thickness, whereas family history of cardiovascular disease was unrelated.

Solution structure of DNA containing alpha-OH-PdG: the mutagenic adduct produced by acrolein.

Acrolein is a cell metabolic product and a main component of cigarette smoke. Its reaction with DNA produces two guanine lesions gamma-OH-PdG, a major adduct that is nonmutagenic in mammalian cells, and the positional isomer alpha-OH-PdG. We describe here the solution structure of a short DNA duplex containing a single alpha-OH-PdG lesion, as determined by solution NMR spectroscopy and restrained molecular dynamics simulations. The spectroscopic data show a mostly regular right-handed helix, locally perturbed at its center by the presence of the lesion. All undamaged residues of the duplex are in anti orientation, forming standard Watson-Crick base-pair alignments. Duplication of proton signals near the damaged site differentiates two enantiomeric duplexes, thus establishing the exocyclic nature of the lesion. At the lesion site, alpha-OH-PdG rotates to a syn conformation, pairing to its counter cytosine residue that is protonated at pH 5.9. Three-dimensional models produced by restrained molecular dynamics simulations show different hydrogen-bonding patterns between the lesion and its cytosine partner and identify further stabilization of alpha-OH-PdG in a syn conformation by intra-residue hydrogen bonds. We compare the alpha-OH-PdG.dC duplex structure with that of duplexes containing the analogous lesion propano-dG and discuss the implications of our findings for the mutagenic bypass of acrolein lesions.