Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H₂O₂ was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H₂O₂, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.     

DNA adducts in human placenta as biomarkers for environmental pollution, analysed by the 32P-HPLC method

During pregnancy, mothers are exposed to complex chemical mixtures, such as air pollution and smoke from incomplete combustion. In this study DNA adducts were measured in human placentas from 29 mothers. Environmental exposure and several possible biomarkers in relation to levels of DNA adducts were measured. Placental aromatic and bulky DNA adducts were measured with the ³²     

Determination of free concentration of sameridine in blood plasma by ultrafiltration and coupled-column liquid chromatography

Sameridine is a new candidate drug with both local anaesthetic and analgesic properties. The free concentration of sameridine in blood plasma was determined by coupled-column liquid chromatography. Following adjustment of the pH and the temperature of the plasma samples, the free fraction was prepared by ultrafiltration. The coupled-column liquid chromatographic system consisted of a reversed-phase column, a cation-exchange extraction column and a cation-exchange analytical column. Sameridine was detected by UV determination at 205 nm and the system showed high selectivity. The limit of quantification was 1 nM and the within-day precision was 4.6% (R.S.D., n=10).     

Identification of two imidazole binding sites and key residues for substrate specificity in human primary amine oxidase AOC3

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 ? sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 ? structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.     

Influence of relative humidity on analyzing electric field exposure using ELF electric field measurements

The objective of the study was to investigate the influence of humidity on analyzing electric field exposure using extremely low frequency (ELF) electric field measurements. The study included 322 measurements in a climate room. We used two commercial three‐axis meters, EFA‐3 and EFA‐300, and employed two measurement techniques in the climate room where we varied the temperature from 15 to 25 °C, the relative humidity from 55% to 95%, and the electric field from 1 to 25 kV/m. We calculated Pearson correlations between humidity and percentage errors for all data and for data at different levels of humidity. When the relative humidity was below 70%, the results obtained by the different measurement methods in terms of percentage errors were of the same order of magnitude for the considered temperatures and field strength, but the results were less reliable when the relative humidity was higher than 80%. In the future, it is important to take humidity into account when electric field measurement results will be compared to the values given in different exposure guidelines. Bioelectromagnetics 34:414–418, 2013. © 2012 Wiley Periodicals, Inc.     

Numerical evaluation of currents induced in a worker by ELF non‐uniform electric fields in high voltage substations and comparison with experimental results

An ungrounded human, such as a substation worker, receives contact currents when touching a grounded object in electric fields. In this article, contact currents and internal electric fields induced in the human when exposed to non‐uniform electric fields at 50 Hz are numerically calculated. This is done using a realistic human model standing at a distance of 0.1–0.5 m from the grounded conductive object. We found that the relationship between the external electric field strength and the contact current obtained by calculation is in good agreement with previous measurements. Calculated results show that the contact currents largely depend on the distance, and that the induced electric fields in the tissues are proportional to the contact current regardless of the non‐uniformity of the external electric field. Therefore, it is concluded that the contact current, rather than the spatial average of the external electric field, is more suitable for evaluating electric field dosimetry of tissues. The maximum induced electric field appears in the spinal cord in the central nervous system tissues, with the induced electric field in the spinal cord approaching the basic restriction (100 mV/m) of the new 2010 International Commission on Non‐Ionizing Radiation Protection guidelines for occupational exposure, if the contact current is 0.5 mA. Bioelectromagnetics 34:61–73, 2013. © 2012 Wiley Periodicals, Inc.     

Occupational exposure to electric fields and induced currents associated with 400 kV substation tasks from different service platforms

The aim of the study was to investigate the occupational exposure to electric fields, average current densities, and average total contact currents at 400 kV substation tasks from different service platforms (main transformer inspection, maintenance of operating device of disconnector, maintenance of operating device of circuit breaker). The average values are calculated over measured periods (about 2.5 min). In many work tasks, the maximum electric field strengths exceeded the action values proposed in the EU Directive 2004/40/EC, but the average electric fields (0.2–24.5 kV/m) were at least 40% lower than the maximum values. The average current densities were 0.1–2.3 mA/m² and the average total contact currents 2.0–143.2 µA, that is, clearly less than the limit values of the EU Directive. The average values of the currents in head and contact currents were 16–68% lower than the maximum values when we compared the average value from all cases in the same substation. In the future it is important to pay attention to the fact that the action and limit values of the EU Directive differ significantly. It is also important to take into account that generally, the workers' exposure to the electric fields, current densities, and total contact currents are obviously lower if we use the average values from a certain measured time period (e.g., 2.5 min) than in the case where exposure is defined with only the help of the maximum values. Bioelectromagnetics 32:79–83, 2011. © 2010 Wiley‐Liss, Inc.     

Occupational exposure to electric fields and currents associated with 110 kV substation tasks

The main aim of this study was to investigate occupational exposure to electric fields, and current densities and contact currents associated with tasks at air-insulated 110 kV substations and analyze if the action value of EU Directive 2004/40/EC was exceeded. Four workers volunteered to simulate the following tasks: Task (A) maintenance of an operating device of a disconnector at ground or floor level, Task (B) maintenance of an operating device of a circuit breaker at ground or floor level, Task (C) breaker head maintenance from a man hoist, and Task (D) maintenance of an operating device of a circuit breaker from a service platform. The highest maximum average current density in the neck was 1.8 mA/m(2) (calculated internal electric field 9.0-18.0 mV/m) and the highest contact current was 79.4 μA. All measured values at substations were lower than the limit value (10 mA/m(2)) of the EU Directive 2004/40/EC and the 2010 basic restrictions (0.1 and 0.8 V/m for central nervous system tissues of the head, and all tissues of the head and body, respectively) of the International Commission on Non-Ionizing Radiation Protection (ICNIRP).     

Competition between CTL narrows the immune response induced by prime-boost vaccination protocols

Recombinant vaccines encoding strings of virus- or tumor-derived peptides and/or proteins are currently being designed for use against both cancer and infectious diseases. These vaccines aim to induce cytotoxic immune responses against several Ags simultaneously. We developed a novel tetramer-based technique, based on chimeric HLA A2/H-2K(b) H chains, to directly monitor the CTL response to such vaccines in HLA-A2 transgenic mice. We found that priming and boosting with the same polyepitope construct induced immune responses that were dominated by CTL of a single specificity. When a mixture of viruses encoding single proteins was used to boost the polyepitope primed response, CTL of multiple specificities were simultaneously expanded to highly effective levels in vivo. In addition, we show that a preexisting response to one of the epitopes encoded within a polyepitope construct significantly impaired the ability of the vaccine to expand CTL of other specificities. Our findings define a novel vaccination strategy optimized for the induction of an effective polyvalent cytotoxic response.