Oxidative DNA damage and influence of genetic polymorphisms among urban and rural schoolchildren exposed to benzene

Traffic related urban air pollution is a major environmental health problem in many large cities. Children living in urban areas are exposed to benzene and other toxic pollutants simultaneously on a regular basis. Assessment of benzene exposure and oxidative DNA damage in schoolchildren in Bangkok compared with the rural schoolchildren was studied through the use of biomarkers.Benzene levels in ambient air at the roadside adjacent to Bangkok schools was 3.95-fold greater than that of rural school areas. Personal exposure to benzene in Bangkok schoolchildren was 3.04-fold higher than that in the rural schoolchildren. Blood benzene, urinary benzene and urinary muconic acid (MA) levels were significantly higher in the Bangkok schoolchildren. A significantly higher level of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in leukocytes and in urine was found in Bangkok children than in the rural children. There was a significant correlation between individual benzene exposure level and blood benzene (r_s = 0.193, P < 0.05), urinary benzene (r_s = 0.298, P < 0.05), urinary MA (r_s = 0.348, P < 0.01), and 8-OHdG in leukocyte (r_s = 0.130, P < 0.05). In addition, a significant correlation between urinary MA and 8-OHdG in leukocytes (r_s = 0.241, P < 0.05) was also found. Polymorphisms of various xenobiotic metabolizing genes responsible for susceptibility to benzene toxicity have been studied; however only the GSTM1 genotypes had a significant effect on urinary MA excretion.Our data indicates that children living in the areas of high traffic density are exposed to a higher level of benzene than those living in rural areas. Exposure to higher level of benzene in urban children may contribute to oxidative DNA damage, suggesting an increased health risk from traffic benzene emission.     

Structure of B-MLV capsid amino-terminal domain reveals key features of viral tropism, gag assembly and core formation

The Gag polyprotein is the major structural protein found in all classes of retroviruses. Interactions between Gag molecules control key events at several stages in the cycle of infection. In particular, the capsid (CA) domain of Gag mediates many of the protein-protein interactions that drive retrovirus assembly, maturation and disassembly. Moreover, in murine leukaemia virus (MLV), sequence variation in CA confers N and B tropism that determines susceptibility to the intracellular restriction factors Fv1n and Fv1b. We have determined the structure of the N-terminal domain (NtD) of CA from B-tropic MLV. A comparison of this structure with that of the NtD of CA from N-tropic MLV reveals that although the crystals belong to different space groups, CA monomers are packed with the same P6 hexagonal arrangement. Moreover, interhexamer crystal contacts between residues located at the periphery of the discs are conserved, indicating that switching of tropism does not result in large differences in the backbone conformation, nor does it alter the quaternary arrangement of the disc. We have also examined crystals of the N-tropic MLV CA containing both N- and C-terminal domains. In this case, the NtD hexamer is still present; however, the interhexamer spacing is increased and the conserved interhexamer contacts are absent. Investigation into the effects of mutation of residues that mediate interhexamer contacts reveals that amino acid substitutions at these positions cause severe defects in viral assembly, budding and Gag processing. Based on our crystal structures and mutational analysis, we propose that in MLV, interactions between the NtDs of CA are required for packing of Gag molecules in the early part of immature particle assembly. Moreover, we present a model where proteolytic cleavage at maturation results in migration of CA C-terminal domains into interstitial spaces between NtD hexamers. As a result, NtD-mediated interhexamer contacts present in the immature particle are displaced and the less densely packed lattice with increased hexamer-hexamer spacing characteristic of the viral core is produced.     

EB病毒膜抗原与系统性红斑狼疮相关性的实验研究

目的:探讨EB病毒膜抗原(MA)的表达与系统性红斑狼疮的关系.方法:用直接免疫荧光抗体法(DFA)检测MA在转基因小鼠体内的表达,用间接免疫荧光法(IFA)和免疫印迹法(IBT)分别检测血清中的抗核抗体(ANA)和抗ENA抗体,用Bradford法检测小鼠24h尿蛋白浓度.常规病理HE染色观察小鼠的肾脏病理改变,冰冻切片免疫荧光检测肾脏是否有免疫复合物沉积.结果:表达MA的转基因小鼠组,有8只小鼠ANA阳性,5只小鼠抗ENA抗体阳性,24h尿蛋白浓度平均值为4153.8 g/ml,小鼠肾脏存在多种病理改变及大量免疫复合物在肾小球沉积.对照组小鼠ANA、抗ENA抗体均为阴性,24h尿蛋白浓度平均值为382.7g/ml,肾脏未见病理改变.结论:EB病毒MA的表达可能引发SLE.     

The effects of actinomycin D and cordycepin on neurite formation and acetylcholinesterase activity in mouse neuroblastoma cells

Actinomycin D (actD) (0.003–0.10 μg/ml) and cordycepin (3–30 μg/ml) were used to examine the requirement of de novo RNA synthesis in the pH 6.6-induced expression of neurites and acetylcholinesterase activity in C-1300 mouse neuroblastoma cells. ActD at 0.03 and 0.10 μg/ml caused a pronounced stimulation in neurite formation following 20 h of treatment, although by 30 h exposure to actD (0.01–0.10 μg/ml), neurite formation had rapidly declined. Cordycepin (3–30 μg/ml) also inhibited neurite formation in a concentration- and time-dependent manner, although it did not produce an initial stimulation in neurite formation. The pH 6.6-induced increase in acetylcholinesterase activity was inhibited by both actD and cordycepin in a concentration- and time-dependent manner. Cell viabilities in the presence of actD and cordycepin were 90% or greater throughout the course of these studies.The effects of actD on [³H]uridine and [³H]leucine transport into cells and on incorporation into acid-insoluble material showed that actD inhibited RNA synthesis to a greater extent than it inhibited protein synthesis. Cordycepin caused only minor effects on [³H]uridine and [³H]leucine transport into cells and incorporation into acid-insoluble material; these effects were variable and neither concentration- nor time-dependent. The results of this study show that actD can inhibit the pH 6.6-induced expression of neurites and acetylcholinesterase activity in mouse neuroblastoma cells at concentrations which were relatively non-toxic and which caused a greater inhibition of RNA synthesis than of protein synthesis. This suggests that de novo RNA synthesis is required for the expression and maintenance of neurites and acetylcholinesterase activity in mouse neuroblastoma cells. Experiments with cordycepin were consistent with this conclusion.     

Reduction and Subsequent Binding of Ruthenium Ions Catalyzed by Subcellular Components

The reduction of Cl(NH₃)₅Ru(III) and subsequent binding of heterocyclic ligands by the resultant (H₂O)(NH₃)₅Ru(II) ion is shown to be catalyzed by components of rat-liver cells. The presence of air significantly decreases the rate of heterocyclic ligand binding. In the case of microsome and soluble component catalysis, this is probably due to oxidation of the Ru(II) ion prior to complexation. Various inhibitors of electron-transfer proteins were employed in an effort to determine the preferred reducing species. These results lend support to the hypothesis that the antitumor activity of acido ruthenium(III) ammine complexes involves activation by reduction in vivo prior to metal coordination to nucleic acids. Anticancer drugs functioning by this mechanism may be preferentially toxic to or may localize in hypoxic areas of tumors.     

The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2‐[bis(2‐hydroxyethyl)amino]‐2‐(hydroxymethyl)propane‐1,3‐diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site‐directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three‐dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.     

High resolution array in the clinical approach to chromosomal phenotypes

Array genomic hybridization (AGH) has recently been implemented as a diagnostic tool for the detection of submicroscopic copy number variants (CNVs) in patients with developmental disorders. However, there is no consensus regarding the choice of the platform, the minimal resolution needed and systematic interpretation of CNVs. We report our experience in the clinical diagnostic use of high resolution AGH up to 100 kb on 131 patients with chromosomal phenotypes but previously normal karyotype. We evaluated the usefulness in our clinics and laboratories by the detection rate of causal CNVs and CNVs of unknown clinical significance and to what extent their interpretation would challenge the systematic use of high-resolution arrays in clinical application. Prioritizing phenotype-genotype correlation in our interpretation strategy to criteria previously described, we identified 33 (25.2%) potentially pathogenic aberrations. 16 aberrations were confirmed pathogenic (16.4% syndromic, 8.5% non-syndromic patients); 9 were new and individual aberrations, 3 of them were pathogenic although inherited and one is as small as approx 200 kb. 13 of 16 further CNVs of unknown significance were classified likely benign, for 3 the significance remained unclear. High resolution array allows the detection of up to 12.2% of pathogenic aberrations in a diagnostic clinical setting. Although the majority of aberrations are larger, the detection of small causal aberrations may be relevant for family counseling. The number of remaining unclear CNVs is limited. Careful phenotype-genotype correlations of the individual CNVs and clinical features are challenging but remain a hallmark for CNV interpretation.