Characterization of the interaction between the tumour suppressor p53 and heme and its role in the protein conformational dynamics studied by various spectroscopic techniques and hydrogen/deuterium exchange coupled with mass spectrometry.

The tumour suppressor p53 regulates the expression of a myriad of proteins that are important for numerous cellular processes, including apoptosis, cell cycle arrest, DNA repair, metabolism, and even autophagy and ferroptosis. Aside from DNA, p53 can interact with many types of partners including proteins and small organic molecules. The ability of p53 to interact with heme has been reported so far. In this study, we used various spectroscopic studies to conduct a thorough biophysical characterization of the interaction between p53 and heme concerning the oxidation, spin, coordination, and ligand state of heme iron. We found that the p53 oligomeric state and zinc biding ability are preserved upon the interaction with heme. Moreover, we described the effect of heme binding on the conformational dynamics of p53 by hydrogen/deuterium exchange coupled with mass spectrometry. Specifically, the conformational flexibility of p53 is significantly increased upon interaction with heme, while its affinity to a specific DNA sequence is reduced by heme. The inhibitory effect of DNA binding by heme is partially reversible. We discuss the potential heme binding sites in p53 with respect to the observed conformational dynamics changes and perturbed DNA-binding ability of p53 upon interaction with heme.

Ultra-sensitive speciation analysis of tellurium by manganese and iron assisted photochemical vapor generation coupled to ICP-MS/MS.

Photochemical vapor generation (PVG) of Te4+ was undertaken with a simple reactor consisting of a polytetrafluoroethylene reaction coil wrapped around a low-pressure mercury tube lamp and using a flow-injection for sample delivery. The composition of a reaction medium, the influence of irradiation time and the effect of added sensitizers and interferents were investigated using high-resolution continuum source atomic absorption spectrometry and a miniature diffusion flame atomizer. A mixture of 5 M acetic acid and 3.5 M formic acid and sample flow rate of 4 mL min-1 permitting a 36 s irradiation time were found optimal for PVG of Te4+. The addition of 250 mg L-1 Mn2+ and 15 mg L-1 Fe2+ ions as sensitizers enhanced the overall PVG efficiency 2.75-fold to 50 ± 2%. In order to achieve higher sensitivity necessary for determination of Te in real environmental samples, PVG was coupled to inductively coupled plasma triple quadrupole mass spectrometer and detection was performed with O2 in the reaction cell utilizing a mass shift mode of measurement (m/z 128 → m/z 144) to ensure interference free ion detection. A limit of detection 1.3 ng L-1 and repeatability (RSD) 0.9% at 250 ng L-1 were achieved. This ultrasensitive methodology was validated for speciation analysis of Te in water samples of various matrix complexities (fresh water, well water, seawater and contaminated water). Since no response was observed from Te6+ under optimal PVG conditions, Te4+ was selectively determined by direct PVG. The sum of Te4+ and Te6+ was determined after pre-reduction of Te6+ in 6 M HCl (95 °C), evaporation to dryness and reconstitution in the reaction medium containing sensitizers. Very good accuracy was demonstrated by spiked recoveries for both Te4+ and total Te in water samples and also by total Te determination in fresh water Standard Reference Material NIST 1643f.

Longitudinally monitored lifetime changes in blood heavy metal concentrations and their health effects in urban birds.

Urban heavy metal pollution can impair the health of humans and other organisms inhabiting cities. While birds are suggested as one of the appropriate bioindicators for essential and non-essential trace element monitoring, the process of particular elements' accumulation in blood and its possible adverse health effects during ageing of individuals remain unexplored. We have investigated lifetime changes in blood lead (Pb), cadmium (Cd), arsenic (As) and zinc (Zn) concentrations and searched for links to health-related traits in sub-urban free-living great tit (Parus major) population monitored over a long period of time. The blood As concentrations were under the limit of detection in most samples. The blood Pb levels showed a non-linear relationship to individuals age, where the highest Pb concentrations were measured in nestlings and in a very small group of highly senescent birds (over 7 years old), while no clear trend was observed for the majority of the adult age stages. No age-related patterns were found for blood Cd or Zn concentrations. The positive relationship between date of capture and blood Cd and Zn levels may reflect seasonal changes in diet composition. We did not reveal any anaemia-like conditions (decreased total erythrocyte count or increased immature erythrocyte count) in relation to blood heavy metal concentrations in the investigated birds. Total leukocyte counts, heterophil/lymphocyte (H/L) ratio and total heterophil and lymphocyte counts increased with increasing Pb, Cd and Zn concentrations in blood. This study demonstrates the suitability of avian blood for actual heavy metal spatial and temporal biomonitoring even in situations when the precise age of the individuals remains unknown.

Rapid determination of majority cations in yoghurts using on-line connection of capillary electrophoresis with mini-dialysis.

An analyser was constructed on the basis of on-line connection of capillary electrophoresis over a short separation path with continuous mini-dialysis sample collection. The developed instrument was employed for simultaneous determination of the majority minerals K+, Ca2+, Na+ and Mg2+ (and possibly NH4+ ions) in commercially available unflavoured yoghurts. The cations are released from the organic structures by digestion with boiling 6 mol/L HCl. They were separated from residues of the organic matrix by a dialysis probe and were transferred to a stream of water. From the continuous stream, the dialysate was injected into the separation capillary through a flow-gating interface. Within the reliability interval, the determined total mineral content was equal to their contents stated on the yoghurt labels and the content determined by flame atomic absorption spectrometry and complexometric titration. The relative standard deviation of the electrophoretic determination is mostly about 5%.

Associations of urban environmental pollution with health-related physiological traits in a free-living bird species.

Urban environmental pollution results in contamination of the tissues of synanthropic organisms by toxic trace elements with potential impacts on human health. Passerine birds may serve as convenient indicators of such contamination. In this study we investigated the effect of blood and plumage contamination with heavy metals (lead Pb, cadmium Cd, copper Cu, chromium Cr) and arsenic metalloid (As) on condition, health and ornamental colour in free-living great tit (Parus major) males from 13 cities across the Czech Republic (EU), mist netted during the early breading season (April-May). Our results showed a significant association of heavy metal tissue contamination with immune function, namely leukocyte composition in the avian blood circulation. High heavy metal contamination in bird feathers was linked to a high heterophil/lymphocyte (H/L) ratio, indicating long-term stress in individuals inhabiting heavily polluted environments. In contrast, males with higher concentrations of heavy metals in blood had a lower H/L ratio, assumingly due to the direct toxicity of heavy metals in certain cell types. This is also supported by traits indicative of anaemia-like haemolytic conditions (decreased absolute erythrocyte count) and increased haematopoiesis (a tendency for increased frequencies of immature erythrocytes). We did not find any association of heavy metal contamination with the bacteriolytic activity of plasma complement, feather growth or ornamentation (black breast stripe area and yellow colouration). There was no significant relationship between heavy metal contamination in blood or feathers and PM10 pollution at the study sites. Our correlational study is the first to show on a large geographic scale that despite strict European air pollution regulations and regular monitoring that have allowed general improvements in atmospheric contamination, non-degradable heavy metals persistently contaminate animal blood and feathers in anthropogenic environments at levels that may have subclinical yet physiological effects with varied influence on health.

Determination of dasatinib in the tablet dosage form by ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis.

Dasatinib is a novel oral prescription drug proposed for treating adult patients with chronic myeloid leukemia. Three analytical methods, namely ultra high performance liquid chromatography, capillary zone electrophoresis, and sequential injection analysis, were developed, validated, and compared for determination of the drug in the tablet dosage form. The total analysis time of optimized ultra high performance liquid chromatography and capillary zone electrophoresis methods was 2.0 and 2.2 min, respectively. Direct ultraviolet detection with detection wavelength of 322 nm was employed in both cases. The optimized sequential injection analysis method was based on spectrophotometric detection of dasatinib after a simple colorimetric reaction with folin ciocalteau reagent forming a blue-colored complex with an absorbance maximum at 745 nm. The total analysis time was 2.5 min. The ultra high performance liquid chromatography method provided the lowest detection and quantitation limits and the most precise and accurate results. All three newly developed methods were demonstrated to be specific, linear, sensitive, precise, and accurate, providing results satisfactorily meeting the requirements of the pharmaceutical industry, and can be employed for the routine determination of the active pharmaceutical ingredient in the tablet dosage form.

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity.

The globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction's kinetics. The kcat values of the heme Fe(III)-OH(-), Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1-1.2 min(-1), and their Km(ATP) values were 18.9-35.4 µM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min(-1) but a very high Km(ATP) value of 357 µM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and Km(ATP) values of 0.4 min(-1) and 78 µM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and Km(ATP) values of 0.3 min(-1) and 33.6 µM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme's catalytic activity because they modulate its ATP binding affinity and thus change its kcat/Km(ATP) value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed.