Using of chicken antibodies for metallothionein detection in human blood serum and cadmium‐treated tumour cell lines after dot‐ and electroblotting

Metallothionein (MT) is a low‐molecular mass protein playing an essential role in homeostasis of heavy metal ions. Its relation with formation and progression of a tumour disease is discussed in this article. Here, we propose a new methodological approach for visualization of MT on PVDF membranes after dot‐ and electroblotting by using a commercial mouse monoclonal antibody E9 and polyclonal chicken antibodies. The optimized procedure was as follows. We dotted 1 μL sample volume on PVDF membrane and let it to dry. Then, we blocked the membrane surface with 2% BSA in PBS for 30 min. After that, the membrane was incubated in chicken primary antibody (diluted 1:500), washed, and incubated in rabbit‐anti‐chicken secondary antibody conjugated with horseradish peroxidase. To visualize the interaction, we used 3‐aminoethyl‐9‐carbazole. Under these conditions, we estimated detection limit as 3 pg of MT per 1 μL. The optimal approach was further utilized for detection of MT level in two human fibroblast cell lines and in blood serum obtained from children with medulloblastoma. The results were in good agreement with differential pulse voltammetry‐Brdicka reaction.     

Advances in capillary electrophoretically mediated microanalysis

Recent developments in the use of capillary electrophoretic techniques for the in-line study of enzyme reactions and derivatization protocols are reviewed. The article is divided into two parts: (i) in-line enzyme reactions and (ii) in-line derivatization. The first part introduces electrophoretically mediated microanalysis (EMMA) and discusses and illustrates the different modes of EMMA. A literature overview is provided, starting from 1996, and the investigated enzymes are classified into two tables based on the mode of engagement (i.e., continuous or transient) of the developed EMMA-based assay. The second part starts with an introduction of the procedures and the nomenclature used in the area of in-line derivatization protocols based on EMMA. Reported derivatization procedures are discussed and classified in tables, according to the functional group that is derivatized.     

Inhibition study of angiotensin converting enzyme by capillary electrophoresis after enzymatic reaction at capillary inlet

Capillary electrophoresis was used to study the inhibition of angiotensin-converting enzyme (ACE) by different inhibitors. Reaction occurred at the capillary inlet during a predetermined waiting period, followed by the electrophoretic separation of the reaction compounds. ACE activity was determined by the quantification of the reaction product, hippuric acid, at 230 nm. The technique was used to study the potency of five different inhibitors (captopril, lisinopril, perindoprilat, quinaprilat and benazeprilat). During a kinetic study, the Ki value of captopril was estimated to be 55.4 +/- 8.8 nM, a value consistent with previously reported values.     

Mean life of the 21/2+ isomer of89Zr

The mean life of the lowest 21/2(⁺ level of⁸⁹Zr has been measured with a pulsed⁷Li beam and the⁸⁵Rb(⁷Li, 3n)⁸⁹Zr reaction. The mean life is τ=7.39 ± 0.23 ns and the reduced transition probability to the 17/2⁺ state isB(E2)=73±2e²fm⁴. The comparison with shell-model predictions requires a large value of the effective chargee _p ≈ 2) to account for the experimental result. The mean life of the 13/2^? state has also been measured and results to be τ=3.22±0.17ns.     

Liquid-crystalline supramolecular polymers formed through complementary nucleobase-pair interactions

We report how the placement of nucleobase units, thymine, or N6-(4-methoxybenzoyl)adenine, onto the ends of a mesogenic core, bis-4-alkoxy-substituted bis(phenylethynyl)benzene, affects the properties of these materials. We show that addition of these bulky polar groups significantly reduces the range of liquid-crystalline behavior of these compounds. However, mixing two complementary nucleobase-containing AA- and BB-type monomer units together does result in the formation of stable, thermotropic liquid-crystalline (LC) phases. Hydrogen bonding is shown to play an important role in the formation of these LC phases, consistent with the formation of oligomeric or polymeric hydrogen-bonded aggregates. X-ray analyses of these mixed materials are consistent with the formation of smectic C phases.     

Nucleobases as supramolecular motifs

The five main natural nucleobases adenine, cytosine, guanine, thymine and uracil are involved in the self-assembly of one of nature's most interesting and intriguing class of biopolymers, namely the nucleic acids DNA and RNA. As such, these nucleobases have held a fascination to researchers in a diverse range of fields. With the growth in the field of supramolecular chemistry and consequently a better understanding of how molecules interact with each other, more and more information is emerging on the complex supramolecular behaviour of these nucleobase. This tutorial review tries to bring together some of the basic concepts of how nucleobases can interact not only with each other, but also with other small organic molecules as well as metals and then looks at how such an understanding is starting to influence the development of new materials and polymers.     

Utilization of a combination of weak hydrogen-bonding interactions and phase segregation to yield highly thermosensitive supramolecular polymers

Supramolecular polymerization, i.e., the self-assembly of polymer-like materials through the utilization of the noncovalent bond, is a developing area of research. In this paper, we report the synthesis and investigation of nucleobase-terminated (N6-anisoyl-adenine and N4-(4-tert-butylbenzoyl)cytosine) low molecular weight poly(THF) macromonomers (<2000 g mol(-1)). Even though the degree of interaction between the nucleobase derivatives is very low (<5 M(-1)) these macromonomers self-assemble in the solid state to yield materials with film and fiber-forming capability. While the mechanical properties of films of both materials show extreme temperature sensitivity, resulting in the formation of very low viscosity melts, they do behave differently, which is attributed to the nature of the self-assembly controlled by the nucleobase. A combination of FT-IR, WAXD, and rheological experiments was carried out to further investigate the nature of the self-assembly in these systems. The studies demonstrate that a combination of phase segregation between the hard nucleobase chain ends and the soft poly(THF) core combined with aromatic amide hydrogen bonding is utilized to yield the highly thermosensitive supramolecular polymeric materials. In addition, analysis of the data suggests that the rheological properties of these supramolecular materials is controlled by the disengagement rate of the nucleobase chain ends from the "hard" phase, which, if shown to be general, provides a design criteria in the development of more thermally responsive materials.     

Determination of chromium(VI) and total chromium in water by in-electrode coulometric titration in a porous glassy carbon electrode

Chromium(VI) is determined through its direct electrochemical reduction in the bulk of a porous glassy carbon electrode. An electrode filled with the acidified sample and Cr(VI) is reduced by means of a constant current whereas the potential of the electrode is monitored. The limits of detection and quantification were found to be 1.9 and 6.0 μg · L⁻¹, resp. The linear range, repeatability and reproducibility were found to be 5–500 μg · L⁻¹, 1.2, and 1.8%, resp. The influence of Fe(III), Ca(II), Mg(II), sulphates, nitrates, humic acids and surfactants was investigated. Total chromium was measured after chemical oxidation of Cr(III) to chromate by permanganate. The method was applied to analyses of water samples.     

Employment of Electrochemical Techniques for Metallothionein Determination in Tumor Cell Lines and Patients with a Tumor Disease

In the present paper we employed adsorptive transfer stripping technique coupled with chronopotentiometric stripping analysis for determination of metallothionein (MT) in tumor cell lines and differential pulse voltammetry Brdicka reaction for determination of MT in blood serum of patients with head and neck cancer or retinoblastoma, and of rats treated with cisplatin with respect to discuss the role of MT in formation of resistance on treatment with heavy metal based cytostatics. The cisplatin or carboplatin sensitive and resistant neuroblastoma cell lines were derived from the maternal cell line isolated from the bone metastasis of patients with neuroblastoma. Based on the results obtained it can be concluded that level of MT increases with higher dose of platinum based cytostatics at cells. Further we focused on determination of MT in blood serum of rats treated with cisplatin (two doses 1.05 mg and/or 2.1 mg of cisplatin per kg). The highest level of MT at rats treated with 1.05 mg cisplatin was determined after four hours as 4.9 μmol/L. In the case of the second experimental group the maximum was reached even after two hours of the treatment as 4.8 μmol/L. In addition we were interested in the effect of cisplatin or carboplatin treatment of patients with a tumor disease. At patients with tumor in head and neck area treated with cisplatin we observed that the level of MT was going higher due to administration of the drug. This phenomenon was observed at all patients. However at patients with retinoblastoma treated with carboplatin we observed various phenomena including decreasing, increasing or no changes in MT level. Progression of MT levels was therefore individual and probably depended on tumor resistance to carboplatin.