A simple time-resolved fluorescence assay for quantitative determination of DOTA chelator.

DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) is one of the preeminent metal chelator applied for diagnostic and therapeutic purposes, however to date there is no versatile and reliable nonradioisotopic method for its precise determination. In this technical note, we present a novel and sensitive fluorimetric assay for quantitative determination of DOTA based on the luminescence quenching of the highly luminescent europium ions complex with trioctyl phosphine oxide and naphthoyl trifluoroacetone sensitizing activators. The assay is carried out in two simple steps and enables the determination of DOTA in the nanomolar range providing a superior tool compared to commonly applied spectrophotometric assay with Arsenazo-III reagent.

A simple and reproducible protocol of glass surface silanization for TIRF microscopy imaging.

We describe a simple and reproducible protocol for the preparation of microscope glass slides for in vitro motility assays that use total internal reflection fluorescence microscopy. The developed method utilizes trimethylchlorosilane (TMCS) as a silanizing reagent, which in the presence of imidazole as a catalyst and under optimized conditions enables reproducible preparation of high-quality hydrophobic glass surfaces. This method presents a simplification and improvement in reproducibility over the commonly applied protocol utilizing dichlorodimethylsilane (DDS) as a silanizing agent. We demonstrate the applicability of the new method by performing the analysis of the interactions of a molecular motor, kinesin-1 with microtubules.

tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level.

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward L-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were L-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.

A simple method for simultaneous RP-HPLC determination of indolic compounds related to bacterial biosynthesis of indole-3-acetic acid.

In this short technical report, we present a fast and simple procedure for sample preparation and a single-run Reversed Phase High Performance Liquid Chromatography (RP-HPLC) determination of seven indoles (indole-3-acetic acid, indole-3-acetamide, indole-3-acetonitrile, indole-3-ethanol, indole-3-lactic acid, tryptamine and tryptophan) in bacterial culture supernatants. The separation of the analytes, after a single centrifugal filtration clean-up step, was performed using a gradient elution on a symmetry C8 column followed by fluorimetric detection (λ(ex) = 280/λ(em) = 350 nm). The calibration curves were linear for all of the studied compounds over the concentration range of 0.0625-125 µg mL(-1) (r ( 2 ) ≥ 0.998) and the limits of detection were below 0.015 µg mL(-1). The applicability of the method was confirmed by analysis of Pseudomonas putida culture supernatants.

PSMA targeted conjugates based on dextran.

BACKGROUND: Currently, radiotherapy is one of the most popular choices in clinical practice for the treatment of cancers. While it offers a fantastic means to selectively kill cancer cells, it can come with a host of side effects. To minimize such side effects, and maximize the therapeutic effect of the treatment, we propose the use of targeted radiopharmaceuticals. In the study presented herein, we investigate two synthetic pathways of dextran-based radiocarriers and provide their key chemical and physical properties: stability of the bonding of chelating agent and tertiary structure of obtained formulations and its influence on biological properties. Additionally, PSMA small molecule inhibitor was attached and quantified using DELFIA fluorescence assay. Finally, biological properties and radiolabeling yield were studied using confocal microscopy and ITLC-SG chromatography. RESULTS: Two types of Dex-conjugates - micelle-like nanoparticles (NPs) and non-folded conjugates - were successfully generated and shown to exhibit cellular effects. The tertiary structure of the conjugates was found to influence the selectivity of PSMA and mediate cell binding as well as cellular uptake mechanisms. NPs were shown to be internalized by other, non - PSMA mediated channels. Simultaneously, the uptake of non-folded conjugates required PSMA inhibitor to pass through cell membrane. The radiochemical yield of NHS coupled DOTA chelator was between 91.3 and 97.7% while the TCT-amine bonding showed higher stability and gave the yields of 99.8-100%. CONCLUSIONS: We obtained novel, dextran-based radioconjugates, and presented a superior method of chelator binding, resulting in exquisite radiochemical properties as well as selective cross-membrane transport.

Dietary salicylates in herbs and spices.

The aim of this study was to determine dietary salicylate content in selected culinary herbs and spices, using the RP-HPLC method with fluorescence detection. The highest concentrations of salicylates were found in dried basil and cumin, followed by dried oregano and cloves. Our research contributes to the global database of salicylate content in food products.

Overall Content of Salicylic Acid and Salicylates in Food Available on the European Market.

The study aimed to determine the salicylates content in 112 products available on the European market. Quantitative determination of free and conjugated forms of salicylic acid in food was performed using reversed-phase high-performance liquid chromatography with fluorescence detection. The salicylates contents ranged from 0 to 1675.79 (µg/100 g). The results of this study confirm the presence of salicylates in food products, as well as a broad content diversity of these compounds depending on the species, variety, and method of processing the food items. The results can be very helpful for nutritionists and dieticians in planning low-salicylates or high-salicylates diets.

Chromosomal Localization and Contribution of Three Homoeologous Genes to Biosynthesis of Cytosolic Aspartate Aminotransferase in Common Wheat.

Chromosomal localization of the three homoeologous genes encoding cytosolic aspartate aminotransferase in common wheat (Triticum aestivum cv. Chinese Spring, 2n = 6x = 42, AABBDD) was specified to: 3AL (0.42÷0.61), 3BL (0.38÷0.41) and 3DL (0.23÷0.81) by a comparative zymographic analysis of the enzymatic activities in deletion lines. It was also attempted to precisely explain the nature of the relationship between a number of genes encoding α and ß subunits and a distribution of staining intensity of cytosolic aspartate aminotransferase allozyme activity bands using aneuploid lines of common wheat with modified third pair of homoeologous chromosomes from genomes A, B and D, on which the genes encoding subunit α (genome A) and ß (genome B and D) are localized. The highest consistency between the experimental results and the theoretical distributions was achieved by substituting values of α = 0.57 and ß = 0.43 in a theoretical model. These results demonstrate that the individual participation of the diploid genome A in the biosynthesis of the cytosolic aspartate aminotransferase allozymes subunits is greater than the individual participation of the diploid genomes B and D.

A novel, simple, and sensitive colorimetric method to determine aromatic amino acid aminotransferase activity using the Salkowski reagent.

This study describes the development of a new colorimetric assay to determine aromatic amino acid aminotransferase (ArAT) activity. The assay is based on the transamination of L-tryptophan in the presence of 2-oxoglutarate, which yields indole-3-pyruvate (IPyA). The amount of IPyA formed was quantified by reaction with the Salkowski reagent. Optimized assay conditions are presented for ArAT isozymes isolated from Pseudomonas putida. For comparative purposes, ArAT activity was also determined by high-performance liquid chromatography. ArAT activity staining in polyacrylamide gels with the Salkowski reagent is also presented.

Alterations in cytosolic glucose-phosphate metabolism affect structural features and biochemical properties of starch-related heteroglycans.

The cytosolic pools of glucose-1-phosphate (Glc-1-P) and glucose-6-phosphate are essential intermediates in several biosynthetic paths, including the formation of sucrose and cell wall constituents, and they are also linked to the cytosolic starch-related heteroglycans. In this work, structural features and biochemical properties of starch-related heteroglycans were analyzed as affected by the cytosolic glucose monophosphate metabolism using both source and sink organs from wild-type and various transgenic potato (Solanum tuberosum) plants. In leaves, increased levels of the cytosolic phosphoglucomutase (cPGM) did affect the cytosolic heteroglycans, as both the glucosyl content and the size distribution were diminished. By contrast, underexpression of cPGM resulted in an unchanged size distribution and an unaltered or even increased glucosyl content of the heteroglycans. Heteroglycans prepared from potato tubers were found to be similar to those from leaves but were not significantly affected by the level of cPGM activity. However, external glucose or Glc-1-P exerted entirely different effects on the cytosolic heteroglycans when added to tuber discs. Glucose was directed mainly toward starch and cell wall material, but incorporation into the constituents of the cytosolic heteroglycans was very low and roughly reflected the relative monomeric abundance. By contrast, Glc-1-P was selectively taken up by the tuber discs and resulted in a fast increase in the glucosyl content of the heteroglycans that quantitatively reflected the level of the cytosolic phosphorylase activity. Based on (14)C labeling experiments, we propose that in the cytosol, glucose and Glc-1-P are metabolized by largely separated paths.