A Novel Conductometric Urea Biosensor with Improved Analytical Characteristic Based on Recombinant Urease Adsorbed on Nanoparticle of Silicalite

Development of a conductometric biosensor for the urea detection has been reported. It was created using a non-typical method of the recombinant urease immobilization via adsorption on nanoporous particles of silicalite. It should be noted that this biosensor has a number of advantages, such as simple and fast performance, the absence of toxic compounds during biosensor preparation, and high reproducibility (RSD = 5.1 %). The linear range of urea determination by using the biosensor was 0.05–15 mM, and a lower limit of urea detection was 20 μM. The bioselective element was found to be stable for 19 days. The characteristics of recombinant urease-based biomembranes, such as dependence of responses on the protein and ion concentrations, were investigated. It is shown that the developed biosensor can be successfully used for the urea analysis during renal dialysis

Selective Amplification of SPR Biosensor Signal for Recognition of rpoB Gene Fragments by Use of Gold Nanoparticles Modified by Thiolated DNA

Abstract An experimental approach for improving the sensitivity of the surface plasmon resonance (SPR) DNA hybridization sensor using gold nanoparticles (GNPs), modified by specific oligonucleotides, was elaborated. An influence of the ionic strength on the aggregation stability of unmodified GNPs and GNPs modified by the thiolated oligonucleotides was investigated by monitoring a value of light extinction at 520 nm that can be considered as a measure of a quantity of the non-aggregated GNPs. While the unmodified GNPs started to aggregate in 0.2 × saline-sodium citrate (SSC), GNPs modified by the negatively charged oligonucleotides were more stable at increasing ionic strength up to 0.5 × SSC. A bioselective element of the SPR DNA hybridization sensor was formed by immobilization on the gold sensor surface of the thiolated oligonucleotides P2, the sequence of which is a fragment of the rpoB gene of Mycobacterium tuberculosis. The injections into the measuring flow cell of the SPR spectrometer of various concentrations of GNPs modified by the complementary oligonucleotides T2-18m caused the pronounced concentration-dependent sequence-specific sensor responses. The magnitude of the sensor responses was much higher than in the case of the free standing complementary oligonucleotides. According to the obtained experimental data, the usage of GNPs modified by specific oligonucleotides can amplify the sensor response of the SPR DNA hybridization sensor in ~1200 times

Transcriptional and post-transcriptional control of eEF1A2 expression during myoblast diffrerentiation

During postnatal development, the switch of the expression from isoform A1 to the isoform A2 of eukaryotic translation elongation factor (eEF1A) is observed in neuronal and muscle tissues. The switch of the expression is a vital fundamental process, as mutant mice, with the partial EEF1A2 deletion dies on the 28th day after birth. Mechanism of the inhibition of A1 and stimulation of A2 expression during the first days of postnatal development is unknown. The existence of potential miRNA binding sites in the 3’UTR of mRNAs encoding the isoforms assumes a post-transcriptional control of abovementioned phenomenon. Aim. To check the possibility of post-transcriptional regulation of the isoforms A1 and A2 expression during differentiation of the human immortalized myoblasts cell line LHCN. Methods. The level of gene expression was quantified by qPCR, the existence of post-transcriptional regulation was demonstrated with Dual-Luciferase® Reporter Assay. Results. Using immortalized human myoblasts cell line LHCN, the induction of isoform A2 of eEF1 during differentiation of myoblasts was shown. The existence of transcriptional and post-transcriptional control of the abovementioned process was confirmed. Downregulation of mir-661 and mir-744 that have binding sites in the 3’ UTR of EEF1A2 mRNA, during differentiation suggests a potential role of microRNAs in the eEF1A2 induction during myoblast differentiation. Conclusions. Induction of A2 isoform of eEF1 during differentiation of myoblasts occurs on transcriptional and post-transcriptional level

PTI-1: novel way to oncogenicity

Aim. The prostate tumor-inducing oncogene (PTI-1), presumably encoding a truncated form of eukaryotic translation elongation factor 1A1 (eEF1A1), was discovered as a gene overexpressed in prostate tumor samples and absent in normal tissues. The mechanism of PTI-1 oncogenicity remains obscure. Methods. Several bioinformatics methods were applied to analyze the PTI-1 mRNA structure, translation efficiency and coding potential. Results. In silico analysis of 5'UTR of its mRNA suggest that PTI-1 mRNA most probably belongs to the class of templates with low translation efficiency. Additionally, novel open reading frame (ORF) starting with alternati- ve initiation site situated upstream of the main ORF start codon was found. Finally, the peptide that does not resemble eEF1A1 but is partially homologous to relaxin can be synthesized. Conclusions. We suggest that the alternative upstream start codon may initiate synthesis of a peptide (uPTI-1) homologous to relaxin, the hormone shown to promote the prostate cancer progression. uPTI-1 protein may interact with the respective relaxin-specific receptors, suggesting that the tumorigenic outcome of PTI-1 is possibly realized via the relaxin-dependent pathway

Polyaniline-coated microtiter plates for use in longwave optical bioassays

A technique for coating the wells of microtiterplates with polyaniline layers and with polyaniline/enzyme layers is presented. The resulting wells are shown to be useful for assaying enzyme substrates (as exemplified for glucose via pH) and hydrogen peroxide (via the redox properties of the film). Analyte detection is based on monitoring the absorption spectra of the polyaniline, which turn purple as a result of redox processes, or green on formation of acids by enzymatic reactions. Hydrogen peroxide (a species produced by all oxidases) and glucose (which yields protons on enzymatic oxidation) have been determined in the millimolar to micromolar concentration range. High sensitivity, film stability and good reproducibility of the measurements make the system an attractive alternative to existing biosensing schemes

Isoforms of elongation factor eEF1A may be differently regulated at post-transcriptional level in breast cancer progression

Eukaryotic translation elongation factor 1A exists as two 98 % homologous isoforms: eEF1A1 (A1) and eEF1A2 (A2) which are tissue and development specific. Despite high homology in an open reading frame (ORF) region, mRNAs coding for eEF1A1 and eEF1A2 are different in their untranslated regions (UTR), suggesting a possibility of their dissimilar post-transcriptional regulation. Aim. To analyze the existence of cis-acting motifs in the UTRs of EEF1A1/A2 mRNAs, to confirm the possibility of post-transcriptional control of eEF1A1 and eEF1A2 expression. Methods. An ensemble of bioinformatic methods was applied to predict regulatory motifs in the UTRs of EEF1A1/A2 mRNAs. Dual-luciferase reporter assay was employed to detect post-transcriptional regulation of eEF1A1/A2 expression. Results. Numerous regulatory motifs in the UTR of EEF1A1/A2 mRNAs were found bioinformatically. The experimental evidence was obtained for the existence of negative regulation of EEF1A1 and positive regulation of EEF1A2 mRNA in the model of breast cancer development. Conclusions. EEF1A1 and EEF1A2 mRNAs contain distinct motifs in the UTRs and are differently regulated in cancer suggesting the possibility of their control by different cellular signals

Colorimetric biomimetic sensor systems based on molecularly imprinted polymer membranes for highly-selective detection of phenol in environmental samples

Aim. Development of an easy-to-use colorimetric sensor system for fast and accurate detection of phenol in envi- ronmental samples. Methods. Technique of molecular imprinting, method of in situ polymerization of molecularly imprinted polymer membranes. Results. The proposed sensor is based on free-standing molecularly imprinted polymer (MIP) membranes, synthesized by in situ polymerization, and having in their structure artificial binding sites capable of selective phenol recognition. The quantitative detection of phenol, selectively adsorbed by the MIP membranes, is based on its reaction with 4-aminoantipyrine, which gives a pink-colored product. The intensity of staining of the MIP membrane is proportional to phenol concentration in the analyzed sample. Phenol can be detected within the range 50 nM–10 mM with limit of detection 50 nM, which corresponds to the concentrations that have to be detected in natural and waste waters in accordance with environmental protection standards. Stability of the MIP-membrane-based sensors was assessed during 12 months storage at room temperature. Conclusions. The sensor system provides highly-selective and sensitive detection of phenol in both mo- del and real (drinking, natural, and waste) water samples. As compared to traditional methods of phenol detection, the proposed system is characterized by simplicity of operation and can be used in non-laboratory conditions

Different Oligomeric Properties and Stability of Highly Homologous A1 and Proto-Oncogenic A2 Variants of Mammalian Translation Elongation Factor eEF1

Translation elongation factor 1A (eEF1A) directs aminoacyl-tRNA to the A site of 80S ribosomes. In addition, more than 97% homologous variants of eEF1A, A1 and A2, whose expression in different tissues is mutually exclusive, may fulfill a number of independent moonlighting functions in the cell; for instance, the unusual appearance of A2 in an A1-expressing tissue was recently linked to the induction of carcinogenesis. The structural background explaining the different functional performance of the highly homologous proteins is unclear. Here, the main difference in the structural properties of these proteins was revealed to be the improved ability of A1 to self-associate, as demonstrated by synchrotron small-angle X-ray scattering (SAXS) and analytical ultracentrifugation. Besides, the SAXS measurements at different urea concentrations revealed the low resistance of the A1 protein to urea. Titration of the proteins by hydrophobic dye 8-anilino-1-naphthalenesulfonate showed that the A1 isoform is more hydrophobic than A2. As the different association properties, lipophilicity, and stability of the highly similar eEF1A variants did not influence considerably their translation functions, at least <i>in vitro</i>, we suggest this difference may indicate a structural background for isoform-specific moonlighting roles