DNA značená aminofenylem a nitrofenylem. Syntéza pomocí enzymatické inkorporace modifikovaných nukleosid trifosfátů, studium elektrochemických vlastností takto značené DNA

We employed single step aqueous phase Suzuki-Miyaura cross-coupling reactions of halogenated 2'-deoxynucleoside 5'-triphosphates with 3-aminophenyl and 3-nitrophenylboronic acid for synthesis of modified nucleoside triphosphates (dNTPs). These dNTPs were then enzymatically incorporated into DNA in Primer extension experiment (PEX). Electrochemical detection using square-wave voltammetry of single-strand modified oligonucleotides (ONs) bearing 3-aminophenyl and 3-nitrophenyl tag demonstated excellent utilization in labeling of DNA

An Efficient Method for the Construction of Functionalized DNA Bearing Amino Acid Groups through Cross-Coupling Reactions of Nucleoside Triphosphates Followed by Primer Extension or PCR

Single-step aqueous cross-coupling reactions of nucleobase-halogenated 2'-deoxynucleosides (8-bromo-2'-deoxyadenosine, 7-iodo-7-deaza-2'-deoxyadenosine, or 5-iodo-2'-deoxy-uridine) or their 5'-triphosphates with 4-boronophenylalanine or 4-ethynylphenylalanine have been developed and used for efficient synthesis of modified 2'-deoxynucleoside triphosphates (dNTPs) bearing amino acid groups. These dNTPs were then tested as substrates for DNA polymerases for construction of functionalized DNA through primer extension and PCR. While 8-substituted adenosine triphosphates were poor substrates for DNA polymerases, the corresponding 7-substituted 7-deazaadenine and 5-substituted uracil nucleotides were efficiently incorporated in place of dATP or dTTP, respectively, by Pwo (Pyrococcus woesei) DNA polymerase. Nucleotides bearing the amino acid connected through the less bulky acetylene linker were incorporated more efficiently than those directly linked through a more bulky phenylene group. In addition, combinations of modified dATPs and dTTPs were incorporated by Pwo polymerase. Novel functionalized DNA duplexes bearing amino acid moieties were prepared by this two-step approach. PCR can be used for amplification of duplexes bearing large number of modifications, while primer extension is suitable for introduction of just one or several modifications in a single DNA strand.publishe

Positive effects of voluntary running on metabolic syndrome-related disorders in non-obese hereditary hypertriacylglycerolemic rats.

While metabolic syndrome is often associated with obesity, 25% of humans suffering from it are not obese and the effect of physical activity remains unclear in such cases. Therefore, we used hereditary hypertriaclyglycerolemic (HHTg) rats as a unique model for studying the effect of spontaneous physical activity [voluntary running (VR)] on metabolic syndrome-related disorders, such as dyslipidemia, in non-obese subjects. Adult HHTg males were fed standard (CD) or high-sucrose (HSD) diets ad libitum for four weeks. Within both dietary groups, some of the rats had free access to a running wheel (CD+VR, HSD+VR), whereas the controls (CD, HSD) had no possibility of extra physical activity. At the end of the four weeks, we measured the effects of VR on various metabolic syndrome-associated parameters: (i) biochemical parameters, (ii) the content and composition of triacylglycerols (TAG), diacylglycerols (DAG), ceramides and membrane phospholipids, and (iii) substrate utilization in brown adipose tissue. In both dietary groups, VR led to various positive effects: reduced epididymal and perirenal fat depots; increased epididymal adipose tissue lipolysis; decreased amounts of serum TAG, non-esterified fatty acids and insulin; a higher insulin sensitivity index. While tissue ceramide content was not affected, decreased TAG accumulation resulted in reduced and modified liver, heart and skeletal muscle DAG. VR also had a beneficial effect on muscle membrane phospholipid composition. In addition, compared with the CD group, the CD+VR rats exhibited increased fatty acid oxidation and protein content in brown adipose tissue. Our results confirm that physical activity in a non-obese model of severe dyslipidemia has many beneficial effects and can even counteract the negative effects of sucrose consumption. Furthermore, they suggest that the mechanism by which these effects are modulated involves a combination of several positive changes in lipid metabolism

Effect of voluntary running on oxidative stress parameters.

<p>The data are expressed as mean ± SEM; n = 6, running intensity = 5–8 km/day. U: One unit of SOD is defined as the amount of the enzyme needed to exhibit 50% dismutation of the superoxide radical. Abbreviations: ANOVA, two-way analysis of variance; CD, control diet; GSH-PX, glutathione peroxidase; HSD, high-sucrose diet; SOD, superoxide dismutase; TBARS, thiobarbituric acid reactive substances; VR, voluntary running.</p><p>Effect of voluntary running on oxidative stress parameters.</p

Effect of voluntary running on ceramide content in tissues.

<p>The data are expressed as mean ± SEM; n = 4, running intensity = 5–8 km/day. Abbreviations: ANOVA, Two-way analysis of variance; CD, control diet; HSD, high-sucrose diet; VR, voluntary running.</p><p>Effect of voluntary running on ceramide content in tissues.</p

Effect of voluntary running on metabolic characteristics of experimental groups.

<p>The data are expressed as mean ± SEM; n = 7, running intensity = 5–8 km/day. Abbreviations: ANOVA, two-way analysis of variance; CD, control diet; EAT, epididymal adipose tissue; HSD, high-sucrose diet; ISI, insulin sensitivity index; NEFA, non-esterified fatty acids; PRAT, perirenal adipose tissue; TAG, triacylglycerols; VR, voluntary running.</p><p>Effect of voluntary running on metabolic characteristics of experimental groups.</p

Effect of voluntary running on the proportion of fatty acids in tissue DAG.

<p>Individual species of DAG were analyzed in the liver, heart and soleus muscle of rats from all groups; in the case of the VR groups the rats were selected on the basis of the intensity of their activity (5–8 km/day). From these results, the percentage proportions of saturated fatty acids (SFA expressed as 18:0 + 16:0), monounsaturated fatty acids (MUFA, 18:1) and polyunsaturated fatty acids (PUFA, 18:2) in tissue DAG were calculated. The data are expressed as mean ± SEM, * significant effect of voluntary running within the dietary groups (CD+VR against CD; HSD+VR against HSD); Two-way ANOVA results: ‡ significant effect of VR, † significant effect of HSD, # significant interaction between VR and HSD; <i>P</i> < 0.05. Abbreviations: DAG, diacylglycerol; HHTg, hereditary hypertriacylglycerolemic rats; HSD, high-sucrose diet; VR, voluntary running.</p

Effect of voluntary running on fatty acid composition of soleus muscle membrane phospholipids.

<p>The data are expressed as mean ± SEM; n = 5, running intensity: 5–8 km/day. Abbreviations: ANOVA, Two-way analysis of variance; CD, control diet; HSD, high-sucrose diet; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; SFA, saturated fatty acids; VR, voluntary running.</p><p>Effect of voluntary running on fatty acid composition of soleus muscle membrane phospholipids.</p