Association between the c.*229C>T polymorphism of the topoisomerase IIb binding protein 1 (TopBP1) gene and breast cancer

Topoisomerase IIb binding protein 1 (TopBP1) is involved in cell survival, DNA replication, DNA damage repair and cell cycle checkpoint control. The biological function of TopBP1 and its close relation with BRCA1 prompted us to investigate whether alterations in the TopBP1 gene can influence the risk of breast cancer. The aim of this study was to examine the association between five polymorphisms (rs185903567, rs116645643, rs115160714, rs116195487, and rs112843513) located in the 30UTR region of the TopBP1 gene and breast cancer risk as well as allele-specific gene expression. Five hundred thirty-four breast cancer patients and 556 population controls were genotyped for these SNPs. Allele-specific Top- BP1 mRNA and protein expressions were determined by using real time PCR and western blotting methods, respectively. Only one SNP (rs115160714) showed an association with breast cancer. Compared to homozygous common allele carriers, heterozygous and homozygous for the T variant had significantly increased risk of breast cancer (adjusted odds ratio = 3.81, 95 % confidence interval: 1.63–8.34, p = 0.001). Mean TopBP1 mRNA and protein expression were higher in the individuals with the CT or TT genotype. There was a significant association between the rs115160714 and tumor grade and stage. Most carriers of minor allele had a high grade (G3) tumors classified as T2-T4N1M0. Our study raises a possibility that a genetic variation of TopBP1 may be implicated in the etiology of breast cancer

The Effects of Cocaine on Different Redox Forms of Cysteine and Homocysteine, and on Labile, Reduced Sulfur in the Rat Plasma Following Active versus Passive Drug Injections

Received: 28 November 2012 / Revised: 19 April 2013 / Accepted: 6 May 2013 / Published online: 16 May 2013 The Author(s) 2013. This article is published with open access at Springerlink.comThe aim of the present studies was to evaluate cocaine-induced changes in the concentrations of different redox forms of cysteine (Cys) and homocysteine (Hcy), and products of anaerobic Cys metabolism, i.e., labile, reduced sulfur (LS) in the rat plasma. The above-mentioned parameters were determined after i.p. acute and subchronic cocaine treatment as well as following i.v. cocaine self-administration using the yoked procedure. Additionally, Cys, Hcy, and LS levels were measured during the 10-day extinction training in rats that underwent i.v. cocaine administration. Acute i.p. cocaine treatment increased the total and protein-bound Hcy contents, decreased LS, and did not change the concentrations of Cys fractions in the rat plasma. In turn, subchronic i.p. cocaine administration significantly increased free Hcy and lowered the total and protein-bound Cys concentrations while LS level was unchanged. Cocaine self-administration enhanced the total and protein-bound Hcy levels, decreased LS content, and did not affect the Cys fractions. On the other hand, yoked cocaine infusions did not alter the concentration of Hcy fractions while decreased the total and protein-bound Cys and LS content. This extinction training resulted in the lack of changes in the examined parameters in rats with a history of cocaine self-administration while in the yoked cocaine group an increase in the plasma free Cys fraction and LS was seen. Our results demonstrate for the first time that cocaine does evoke significant changes in homeostasis of thiol amino acids Cys and Hcy, and in some products of anaerobic Cys metabolism, which are dependent on the way of cocaine administration

Cation exchange HPLC analysis of desmosines in elastin hydrolysates

Desmosine crosslinks are responsible for the elastic properties of connective tissues in lungs and cardiovascular system and are often compromised in disease states. We developed a new, fast, and simple cation exchange HPLC assay for the analysis of desmosine and isodesmosine in animal elastin. The method was validated by determining linearity, accuracy, precision, and desmosines stability and was applied to measure levels of desmosines in porcine and murine organs. The detection and quantification limits were 2 and 4 pmol, respectively. The run-time was 8 min. Our cation exchange column does not separate desmosine and isodesmosine, but their level can be quantified from absorbance at different wavelengths. Using this assay, we found that desmosines levels were significantly lower in elastin isolated from various organs of immunodeficient severe combined immunodeficiency mice compared with wild-type animals. We also found that desmosines levels were lower in lung elastin isolated from hyperhomocysteinemic Pcft−/− mice deficient in intestinal folate transport compared with wild-type Pcft+/+ animals

The use of high-performance liquid chromatography with diode array detector for the determination of sulfide ions in human urine samples using pyrylium salts

Hydrogen sulfide is a toxic gas involved in the regulation of some essential biological processes. A novel, precise, accurate and rapid method based on high-performance liquid chromatography with diode array detection for the determination of sulfide ions in human urine sample is proposed. The method involves the derivatization of sulfide with pyrylium salts – (2,4,6-triphenylpyrylium hydrogensulfate(VI) (L1) and 4-[p-(N,N-dimethylamino)phenyl]-2,6-diphenylpyrylium chlorate(VII) (LN1). The separation occurs on InfinityLab Poroshell 120 EC C18 column using acetonitrile and phosphate buffer as a mobile phase. The detectors utilized a wavelength of 371 or 580 nm. The calibration curves were linear in the range of 2–150 μmol L−1 and 1–50 μmol L−1 for L1 and LN1 derivatives, respectively. The samples were found to be stable from sample collection to final analysis. The method was successfully applied to samples from apparently healthy volunteers

H2S biosynthesis and catabolism: new insights from molecular studies

Hydrogen sulfide (H2S) has profound biological effects within living organisms and is now increasingly being considered alongside other gaseous signalling molecules, such as nitric oxide (NO) and carbon monoxide (CO). Conventional use of pharmacological and molecular approaches has spawned a rapidly growing research field that has identified H2S as playing a functional role in cell-signalling and post-translational modifications. Recently, a number of laboratories have reported the use of siRNA methodologies and genetic mouse models to mimic the loss of function of genes involved in the biosynthesis and degradation of H2S within tissues. Studies utilising these systems are revealing new insights into the biology of H2S within the cardiovascular system, inflammatory disease, and in cell signalling. In light of this work, the current review will describe recent advances in H2S research made possible by the use of molecular approaches and genetic mouse models with perturbed capacities to generate or detoxify physiological levels of H2S gas within tissue

A method for the determination of total and reduced methimazole in various biological samples

A simple, rapid, reproducible and sensitive method based on HPLC with ultraviolet detection was developed for the determination of methimazole (MMI) in animal tissues and plasma samples. Under the optimum experimental conditions, the calibration curves for MMI were linear in the tested range 0.5-20 mg kg(-1) tissue sample (mg l(-1) plasma) with correlation coefficients better than 0.99. The performance of the proposed method was tested for the determination of MMI levels in brain, liver, thyroid gland and plasma of MMI-treated hens, as well as in their eggs and embryos. The proposed method reduces time and simplifies the sample preparation procedure.status: publishe

The chemical derivatization in high performance liquid chromatography

High performance liquid chromatography (HPLC) is a method used to determine inorganic and organic substances in biological samples. Nevertheless, many analytes cannot be detected using HPLC method, because they do not contain a necessary chromophoric or fluophoric groups. Derivatization is the solution of this problem. This process can be defined as a conversion of analyte to corresponding derivative which possesses in its structure a moiety compatible with suitable detector [1, 2]. Reagent responsible for conversion of analyte to a derivative needs to meet a lot of requirements. It needs to be selective e.g. to react only with analysed substances and it should not generate by-products. The derivatization reagent should react rapidly, quantitatively, at lowest possible temperature and weakly pH, and the excess of reagent should be easily removable from reaction medium [1, 3, 5]. The derivatization can be carried out in pre-column, post-column and on-column mode. In the pre-column derivatization, analytes are derivatized before injection on HPLC system, and the reaction products are separated and detected. In the post-column derivatization, the reaction is performed automatically by adding the derivatization reagent after separation but before detection. The third method is based on reaction, which simultaneously proceeds with column separation [2, 3, 5, 6]. The derivatization processes in gas and liquid chromatography are subject matter among researcher from all over the world. The Polish literature has only few review articles on derivatization process in liquid chromatography [2, 4, 55]. The present article reviews derivatization techniques used in HPLC. Derivatization techniques used in gas chromatography are classified due to the chemical nature of derivatization reagent [3, 56]. Our attention is focused on the analyte and derivatization reagent, which can be react with various functional groups such as amino, sulfhydryl, hydroxyl or carboxyl groups, occurring in the examined molecules. By chemically modification compounds into derivatives, they obtain necessary properties for chromatographic separation and accurate analysis