Molecular Modeling of Cytochrome P450 1A1: Enzyme-Substrate Interactions and Substrate Binding Affinities

Abstract

Human cytochrome P450 1A1, which is present in lungs, plays an important role in the metabolic activation of chemical carcinogens, and in particular, is thought to be linked to lung cancer. The mechanism of carcinogenesis is related to the enzyme's ability to oxidize highly toxic compounds, such as polycyclic aromatic hydrocarbons (PAHs), to their carcinogenic derivatives.

In order to better understand P450 1A1 function, a homology model of this enzyme has been constructed. The model has been based on the structure of P450 2C5, the first mammalian P450 to be crystallized. The coordinates of the model have been calculated using a consensus strategy, and the resulting structure has been evaluated with the ProStat and Profiles-3D programs. P450 1A1 substrates, such as benzo[a]pyrene, ethoxyresorufin and methoxyre- sorufin, were then docked into the active site of the model, and key amino acid residues able to interact with the substrate, have been identified. The analysis of enzyme-substrate interactions indicated that hydrophobic interactions are mainly responsible for binding of these substrates in the active site. Moreover, the non-bond enzyme-substrate interaction energy for ethoxyresorufin was lower than that for methoxyresorufin, which is consistent with higher activity of 1A1 towards the former substrate. Key residue Val-382 may play an important role in these interactions. Additionally, we performed binding free energy calculations for the three substrates. The obtained values were similar to those observed experimentally, which suggests that this approach might be useful for prediction of binding constants.     

Helicobacter pylori infection and antioxidants can modulate the genotoxic effects of heterocyclic amines in gastric mucosa cells

Helicobacter pylori (H. pylori) infection plays an important role in gastric carcinogenesis. This bacterium may induce cancer transformation and change the susceptibility of gastric mucosa cells to various exogenous dietary irritants. The aim of the study was to evaluate the influence of H. pylori infection on the reaction of the stomach cells to a genotoxic effect of heterocyclic amines (HCAs). These well-known mutagens are formed during cooking of protein-rich foods, primarily meat. Taking into account that persons consuming a mixed-western diet are exposed to these compound nearly an entire lifetime and more than half of human population is infected with H. pylori, it is important to assess the combined effect of H. pylori infection and HCAs in the context of DNA damage in gastric mucosa cells, which is a prerequisite to cancer transformation. We employed 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) because these substances are present in a great amount in cooked and fried meat. Using alkaline comet assay, we showed that the extent of the DNA damage induced by HCAs was significantly higher in H. pylori infected gastric mucosa cells than in non-infected counterparts. We did not observed any difference in the efficiency of repair of DNA lesions induced by HCAs in both type of cells. Vitamin C reduced the genotoxic effects of HCAs in H. pylori infected and non-infected gastric mucosa cells. Melatonin more effectively decreased DNA damage caused by HCAs in H. pylori infected gastric mucosa cells as compared with control. Our results suggest that H. pylori infection may influence the susceptibility of gastric mucosa cells to HCAs and dietary antioxidative substances, including vitamin C and melatonin may inhibit the genotoxic effects of HCAs on gastric mucosa cells and may reduce the risk of carcinogenesis caused by food borne mutagens and H. pylori infection.     

Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

Abstract

It has been shown for the first time that conformational junction between contiguous right- handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)₁₃ and (dC-dG)₁₆ blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease SI cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)_n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)₁₃ segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)_n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Analysis of Age-Related Global DNA Methylation in Chicken

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.     

The association of insulin resistance with serum osteoprotegerin in obese adolescents

Some data indicate a potential relationship between insulin resistance level and the concentration of osteoprotegerin (OPG) in the body. There have been few studies concerning OPG level and its relationship with insulin resistance and body composition in young people. The aim of this study was to assess serum osteoprotegerin concentration in obese adolescents, and to evaluate its potential association with insulin resistance. Seventy-eight obese adolescents and 20 healthy volunteers aged 12–18 years were recruited in the study. Selected anthropometrical measurements and blood biochemical analyses were performed. Insulin resistance in the participants was evaluated according to the homeostasis model assessment-insulin resistance (HOMA-IR) protocol. Level of OPG was assessed in serum. Obese subjects had significantly higher HOMA-IR indices and OPG levels in serum than the control group. A significant positive correlation between OPG and insulin resistance was found. It was observed that high concentrations of osteoprotegerin are associated with insulin resistance in obese adolescents.     

Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm

We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.     

Development of transgenic strains for the biological control of the Mexican fruit fly, Anastrepha ludens

The Mexican fruit fly, Anastrepha ludens, is a highly significant agricultural pest species that has been genetically transformed with a piggyBac-based transposon vector system using independent vector and transposase helper plasmids. Minimum estimated germ-line transformation frequencies were approximately 13–21% per fertile G₀ individual, similar to previously reported frequencies using single vector-helper plasmids. Two vector constructs were tested with potential importance to transgenic strain development for mexfly biological control. The first allows post-integration stabilization of a transposon-vector by deletion of a terminal sequence necessary for mobilization. The complete pB[L1-EGFP-L2-DsRed-R1] vector was integrated into the Chiapas wild type strain with subsequent deletion of the L2-DsRed-R1 sub-vector carrying the piggyBac 3′ terminal sequence. Quality control tests for three of the stabilization vector lines (previous to stabilization) assessed viability at all life stages, fertility, adult flight ability, and adult male sexual competitiveness. All three transgenic lines were less fit compared to the wild strain by approximately 5–10% in most tests, however, there was no significant difference in sexual competitiveness which is the major prerequisite for optimal strain release. The second vector, pB[XL-EGFP, Asß2-tub-DsRed.T3], has the DsRed.T3 fluorescent protein reporter gene regulated by the A. suspensa Asß2-tubulin promoter, that resulted in testis and sperm-specific DsRed fluorescence in transgenic male mexflies. Fluorescent sperm bundles were unambiguously observed in the spermathecae of non-transgenic females mated to transgenic males. One transgenic line apparently had a male-specific Y-chromosome insertion, having potential use for sexing by fluorescent-embryo sorting. All transgenic lines expressed easily detectable and stable fluorescence in adults allowing their identification after trapping in the field.     

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

5-Taurinomethyluridine (τm⁵U) and 5-taurinomethyl-2-thiouridine (τm⁵s²U) are located at the wobble position of human mitochondrial (hmt) tRNA^Leu(UUR) and tRNA^Lys, respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm⁵U and τm⁵s²U into 17-mers related to the sequence of the anticodon arms hmt-tRNA^Leu(UUR) and hmt-tRNA^Lys, respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF₃) are compatible with the blockage of the canonical monomeric units. The synthesis of τm⁵s²U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.