Expression, Purification and Crystallization of Wheat Profilin (Tri a 12)

Profilin from wheat (Triticum aestivum) has been identified as an allergen (Tri a 12). The recombinant 14 kDa protein was produced in Escherichia coli, purified and crystallized using the hangingdrop vapour-diffusion method. A diffraction-data set was collected in-house from a single crystal to a resolution of 3.3 Å. The crystals belonged to space group P3221, with unit-cell parameters a = b = 58.9 Å, c = 82.5 Å, α = β = 90° and γ = 120°. (doi: 10.5562/cca1790

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Qualitative and quantitative determination of proteins in extracts of some medicinal plants

Introduction. Global consumption of plant protein is increasing and high-fiber plants have health benefits through valuable phytochemicals. Plant proteins serve as an alternative to animal proteins to meet consumer demand on the one hand and reduce the risk of disease on the other. Material and Methods. We performed qualitative and quantitative determination of proteins in extracts by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and total content of proteins by Bradford assay. Results. Applied methods for the qualitative and quantitative determination of proteins in dry extracts of medicinal plants: Agrimonia eupatoria L., Cichorium intybus L., Galium verum L., and Solidago virgaurea L. confirm that proteins were detected in all dry extracts and were determined the total content of them. Conclusion. For all extracts obtained from aerial parts of: Agrimonia eupatoria, Cichorium intybus, Galium verum and Solidago virgaurea we find a low protein concentration, which implies minimizing allergic reactions and intolerance to the extracts studied

Aberrant DNA Methylation of ABC Transporters in Cancer

ATP-binding cassette (ABC) transporters play a crucial role in multidrug resistance (MDR) of cancers. They function as efflux pumps, resulting in limited effectiveness or even failure of therapy. Increasing evidence suggests that ABC transporters are also involved in tumor initiation, progression, and metastasis. Tumors frequently show multiple genetic and epigenetic abnormalities, including changes in histone modification and DNA methylation. Alterations in the DNA methylation status of ABC transporters have been reported for a variety of cancer types. In this review, we outline the current knowledge of DNA methylation of ABC transporters in cancer. We give a brief introduction to structure, function, and gene regulation of ABC transporters that have already been investigated for their DNA methylation status in cancer. After giving an overview of the applied methodologies and the CpGs analyzed, we summarize and discuss the findings on aberrant DNA methylation of ABC transporters by cancer types. We conclude our review with the discussion of the potential to target aberrant DNA methylation of ABC transporters for cancer therapy