Biomedical subjects

A V El'skaya

Publications and source records attributed to A V El'skaya.

At least 19 recordsLinked to original sources

PubMed2005

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline.

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.

Acetylcholine↗

PubMed2004

Chaperone-like activity of mammalian elongation factor eEF1A: renaturation of aminoacyl-tRNA synthetases.

Eukaryotic translational elongation factor eEF1A is known to be responsible for the binding of codon-specific aminoacyl-tRNAs to the ribosome. In this study, we report that in addition to this canonical function, eEF1A is able to promote the renaturation of aminoacyl-tRNA synthetases (ARS) and protect them against denaturation by dilution. The full recovery of the phenylalanyl- (PheRS) and seryl-tRNA synthetase (SerRS) activities was achieved in the presence of 4 microM eEF1A, while bovine serum albumin at similar concentration had no renaturation effect. Remarkably, in vitro renaturation occurs at the molar ratio of eEF1A to ARS equivalent to that found in the cytoplasm of higher eukaryotic cells. The eEF1A.GDP and eEF1A.GTP complexes were shown to be similar in their effect on the phenylalanyl-tRNA synthetase renaturation. Thus, we conclude that the chaperone-like activity of eEF1A might be important for maintaining the enzymes activity in the protein synthesis compartments of mammalian cells.

Amino Acyl-tRNA Synthetases↗

PubMed2002

Extended conformation of mammalian translation elongation factor 1A in solution.

The conformation of mammalian elongation factor eEF1A in solution was examined by the small angle neutron scattering and scanning microcalorimetry. We have found that in contrast to the bacterial analogue the eEF1A molecule has no fixed rigid structure in solution. The radius of gyration of the eEF1A molecule (5.2 nm) is much greater than that of prokaryotic EF1A. The specific heat of denaturation is considerably lower for eEF1A than for EF1A, suggesting that the eEF1A conformation is significantly more disordered. Despite its flexible conformation, eEF1A is found to be highly active in different functional tests. According to the neutron scattering data, eEF1A becomes much more compact in the complex with uncharged tRNA. The absence of a rigid structure and the possibility of large conformational change upon interaction with a partner molecule could be important for eEF1A functioning in channeled protein synthesis and/or for the well-known capability of the protein to interact with different ligands besides the translational components.

Animals↗

PubMed2002

A novel enzyme biosensor for steroidal glycoalkaloids detection based on pH-sensitive field effect transistors.

For the design of a biosensor sensitive to steroidal glycoalkaloids, pH-Sensitive Field Effect Transistors as transducers and immobilised butyrylcholinesterase as a biorecognition element have been used. The total potato glycoalcaloids can be measured by this biosensor in the concentration range 0.5-100 microM with detection limits of 0.5 microM for alpha-chaconine and of 2.0 microM for alpha-solanine and solanidine, respectively. The responses of the developed biosensors were reproducible with a relative standard deviation of about 1.5% and 5% for intra- and inter-sensor responses (both cases, n=10, for an alkaloid concentration of 5 microM), respectively. Moreover, due to the reversibility of the enzyme inhibition, the same sensor chip with immobilised butyrylcholinesterase can be used several times (for at least 100 measurements) after a simple washing by a buffer solution and can be stored at 4 degrees C for at least 3 months without any significant loss of the enzymatic activity.

Alkaloids↗

PubMed2000

Polyaniline-coated microtiter plates for use in longwave optical bioassays.

A technique for coating the wells of microtiter-plates 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.

Aniline Compounds↗

PubMed2000

Development of highly selective and stable potentiometric sensors for formaldehyde determination.

Two types of biosensors selective to formaldehyde have been developed on the basis of pH-sensitive field effect transistor as a transducer. Highly or partially purified alcohol oxidase (AOX) and the permeabilised cells of methylotrophic yeast Hansenula polymorpha (as a source of AOX) have been used as sensitive elements. The response time in steady-state measurement mode is in the range of 10-60 s for the enzyme-based sensors and 60-120 s for the cell-based sensor. When measured in kinetic mode the response time of all biosensors developed was less than 5 s. The linear dynamic range of the sensor output signals corresponds to 5-200 mM formaldehyde for highly and partially purified alcohol oxidase, and 5-50 mM formaldehyde for the cells. The operational stability of the biosensors is not less than 7 h, and the relative standard deviation of intra-sensor response is approximately 2 and 5% for the enzyme- and cell-based sensors, respectively. When stored at 4 degrees C, the enzyme and cell sensor responses have been found stable for more than 60 and 30 days, respectively. Both types of biosensors demonstrate a high selectivity to formaldehyde with no potentiometric response to primary alcohols, including methanol, or glycerol and glucose. The possible reasons of such unexpected high selectivity of AOX-based FET-sensors to formaldehyde are discussed. The influence of the biomembrane composition and the effect of different buffers on the sensor response to formaldehyde are also discussed.

Alcohol Oxidoreductases↗

PubMed1999

Functional interaction of mammalian valyl-tRNA synthetase with elongation factor EF-1alpha in the complex with EF-1H.

In mammalian cells valyl-tRNA synthetase (ValRS) forms a high Mr complex with the four subunits of elongation factor EF-1H. The beta, gamma, and delta subunits, that contribute the guanine nucleotide exchange activity of EF-1H, are tightly associated with the NH2-terminal polypeptide extension of valyl-tRNA synthetase. In this study, we have examined the possibility that the functioning of the companion enzyme EF-1alpha could regulate valyl-tRNA synthetase activity. We show here that the addition of EF-1alpha and GTP in excess in the aminoacylation mixture is accompanied by a 2-fold stimulation of valyl-tRNAVal synthesis catalyzed by the valyl-tRNA synthetase component of the ValRS.EF-1H complex. This effect is not observed in the presence of EF-1alpha and GDP or EF-Tu.GTP and requires association of valyl-tRNA synthetase within the ValRS.EF-1H complex. Since valyl-tRNA synthetase and elongation factor EF-1alpha catalyze two consecutive steps of the in vivo tRNA cycle, aminoacylation and formation of the ternary complex EF-1alpha.GTP. Val-tRNAVal that serves as a vector of tRNA from the synthetase to the ribosome, the data suggest a coordinate regulation of these two successive reactions. The EF-1alpha.GTP-dependent stimulation of valyl-tRNA synthetase activity provides further evidence for tRNA channeling during protein synthesis in mammalian cells.

Acylation↗

PubMed1999

Renaturation of rabbit liver aminoacyl-tRNA synthetases by 80S ribosomes.

Protein biosynthesis machinery is thought to be mostly compartmentalised within the mammalian cell, involving direct interactions between different components of the translation apparatus. The present research concerns the functional meaning of the interaction between the rabbit liver aminoacyl-tRNA synthetases and 80S ribosomes. We have shown that rabbit liver 80S ribosomes are able to enhance the activity of leucyl-tRNA synthetase, which is a component of high-molecular weight aminoacyl-tRNA synthetase complex, and phenylalanyl-tRNA synthetase not associated within this complex. The ribosomes increase the initial rate of both the total reaction of tRNA aminoacylation and the first step of this reaction, the formation of leucyladenylate. Moreover, a positive cooperativity of the tRNA interaction with two binding sites of leucyl-tRNA synthetase is also increased in the presence of highly purified 80S ribosomes. The effect of 80S ribosomes on partly denatured leucyl-tRNA synthetase and phenylalanyl-tRNA synthetase and the protection by 80S ribosomes of both enzymes against inactivation indicate a refolding and stabilising capacity of the ribosomes. It is concluded that the interaction of aminoacyl-tRNA synthetases and 80S ribosomes is important for the maintenance of an active conformation of the enzymes.

Amino Acyl-tRNA Synthetases↗

PubMed1998

An approach to conductometric immunosensor based on phthalocyanine thin film.

A new approach to conductometric biosensors utilizing iodine-sensitive phthalocyanine thin films has been proposed. The excellent sensitivity of the tetra-tert-butyl copper phthalocyanine (ttb-CuPc) to free iodine was used for the first time to detect a peroxidase-initiated reaction in an aqueous medium. To minimize the interfering effect of aqueous electrolytes on the impedance responses of the ttb-CuPc film itself, Au/Cr interdigitated planar electrodes bearing ttb-CuPc thin films were protected with hydrophobic gas-permeable membranes, namely thermally evaporated calixarene or plasma polymerized hexamethyldisiloxane films. Impedance spectroscopy data were analyzed in order to define the optimal operating frequency. An enzyme sensor with peroxidase immobilized in a cross-linked albumin matrix was tested. Its impedance responses were studied under variation of the substrate concentration, pH, ionic strength and buffer capacity. These results were used to define conditions for peroxidase-linked immunoassay in subsequent tests. With the developed sensor, concentrations of IgG in 0.2-2 micrograms/ml range were measured in a competitive mode with satisfactory accuracy. The detection of IgG in both test solutions and blood serum samples has been demonstrated.

Biosensing Techniques↗

PubMed1998

Eukaryotic translation elongation factor 1 alpha: structure, expression, functions, and possible role in aminoacyl-tRNA channeling.

This review offers a comprehensive analysis of eukaryotic translation elongation factor 1 (eEF-1 alpha) in comparison with its bacterial counterpart EF-Tu. Altogether, the data presented indicate some variances in the elongation process in prokaryotes and eukaryotes. The differences may be attributed to translational channeling and compartmentalization of protein synthesis in higher eukaryotic cells. The functional importance of the EF-1 multisubunit complex and expression of its subunits under miscellaneous cellular conditions are reviewed. A number of novel functions of EF-1 alpha, which may contribute to the coordinate regulation of multiple cellular processes including growth, division, and transformation, are characterized.

Animals↗

PubMed1997

Three tRNA binding sites in rabbit liver ribosomes and role of the intrinsic ATPase in 80S ribosomes from higher eukaryotes.

Three tRNA binding sites have been found in organisms of all domains (former kingdoms) with only one exception: Four binding sites have been reported for cytoplasmic 80S ribosomes from rabbit liver. Therefore, the issue was reconsidered, and the data revealed that rabbit liver ribosomes contain three tRNA binding sites, underlining the universal character of this ribosomal feature. Furthermore, a first analysis of the role of the ribosome intrinsic ATPase was performed. This ATPase is found in ribosomes of higher eukarya but not in lower eukarya such as yeast or ribosomes of the domains archea and bacteria. The results suggest that the intrinsic ATPase fulfills the same function as the essential third elongation factor EF-3, an ATPase in higher fungi (yeast etc.), that facilitates the release of the deacylated tRNA from the E site.

Adenosine Triphosphatases↗

PubMed1997

Evidence for the formation of an unusual ternary complex of rabbit liver EF-1alpha with GDP and deacylated tRNA.

Eukaryotic translation elongation factor 1alpha is known to interact in GTP-bound form with aminoacyl-tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF-1alpha*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18-20), has been detected by four independent methods. [EF-1alpha*GDP]-interacting sites are located in the acceptor stem, TpsiC stem and TpsiC loop of tRNA(Phe) and tRNA(Leu) molecules. Both tRNA and EF-1alpha are found to undergo certain conformational changes during their interaction. The ability of EF-1alpha to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl-tRNA channeling in higher eukaryotic cells.

Animals↗

PubMed1994

Thin-film conductometric biosensors for glucose and urea determination.

The characteristics of the developed conductometric biosensors for urea and glucose determination are described. Conductometric transducers based on thin-film interdigitated metal (Au, Cr, Cu, Ni) electrodes were studied, and enzymes urease and glucose oxidase were used for the selective membranes formation on the chips having gold electrodes. The influence of ionic strength and buffer capacity of the samples on the biosensors response in kinetic and steady-state modes of measurements was thoroughly tested. It was shown that the kinetic response of the sensors does not depend on the buffer capacity of the analyzed sample. In basic features the performance of the developed biosensors is rather close to that of respective enzyme field effect transistor, though the former are much superior when the technological complexity of the transducer itself is considered and taking into account that conductometric sensors require no reference electrode.

Biosensing Techniques↗

PubMed1993

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia.

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M(r) 1.82 x 10(6), 0.84 x 10(6) (high-M(r) aminoacyl-tRNA synthetase complexes) and 0.12-0.35 x 10(6). In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82 x 10(6) complex into the 0.84 x 10(6) complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. KM for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivation in vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M(r) complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.

Amino Acyl-tRNA Synthetases↗

PubMed1993

A cell biosensor specific for formaldehyde based on pH-sensitive transistors coupled to methylotrophic yeast cells with genetically adjusted metabolism.

A cell biosensor specific for formaldehyde was developed using double-mutant cells of the methylotrophic yeast Hansenula polymorpha A3-11. The activities of some of the enzymes in the metabolic pathway of the wild-strain cells were deliberately suppressed by introducing respective genetic blocks to optimize the selectivity and acidification rate. Mutant yeast cells produced in this way were immobilized in Ca-alginate gel on the gate of a pH-sensitive field effect transistor. The local acidification of the extracellular medium due to specific conversion of formaldehyde was recorded. The steady-state response time of the biosensor was 2-3 min, i.e., about 10 times shorter than the response time for the alcohol-specific cell biosensors described earlier. The linear dynamic range of the sensor's response corresponds to formaldehyde concentrations of 2 to 200 mM. The operational stability of the sensor was not less than 4 h. The biosensor demonstrated high specificity to formaldehyde with no response to several organic acids, methanol, and other alcohols, except for low sensitivity to ethanol. The influence of sample buffer capacity and pH on the sensor response, as well as thermostability, was investigated.

Biosensing Techniques↗

PubMed1992

A codon sugar structure strongly influences tRNA binding to programmed ribosomes.

To study the role of a codon sugar-phosphate backbone in aminoacyl-tRNA selection on the ribosome a comparison of tRNA(Phe) affinity for pdTpdTpdT and prUprUprU in solution, and for correspondingly programmed 30S ribosomal subunits has been performed. In solution the tRNA(Phe) affinity for pdTpdTpdT appeared to be even slightly higher than for prUprUprU, whereas deoxyribocodon was significantly less efficient in the stimulation of Phe-tRNA(Phe) binding to the 30S ribosomal subunit. Some difference in neomycin action in both systems was revealed.

Anticodon↗

PubMed1991

Interaction of ribo- and deoxyriboanalogs of yeast tRNA(Phe) anticodon arm with programmed small ribosomal subunits of Escherichia coli and rabbit liver.

A synthetic ribooligonucleotide, r(CCAGACUGm-AAGAUCUGG), corresponding to the unmodified yeast tRNA(Phe) anticodon arm is shown to bind to poly(U) programmed small ribosomal subunits of both E. coli and rabbit liver with affinity two order less than that of a natural anticodon arm. Its deoxyriboanalogs d(CCAGACTGAAGATCTGG) and d(CCAGA)r(CUGm-AAGA)d(TCTGG), are used to study the influence of sugar-phosphate modification on the interaction of tRNA with programmed small ribosomal subunits. The deoxyribooligonucleotide is shown to adopt a hairpin structure. Nevertheless, as well as oligonucleotide with deoxyriboses in stem region, it is not able to bind to 30S or 40S ribosomal subunits in the presence of ribo-(poly(U] or deoxyribo-(poly (dT) template. The deoxyribooligonucleotide also has no inhibitory effect on tRNA(Phe) binding to 30S ribosomes at 10-fold excess over tRNA. Neomycin does not influence binding of tRNA anticodon arm analogs used. Complete tRNA molecule and natural modifications of anticodon arm are considered to stabilize the arm structure needed for its interaction with a programmed ribosome.

Animals↗

PubMed1990

Correlation between poly(U) misreading and poly(dT) translation efficiency in E coli cell-free systems.

A positive correlation between poly(U) misreading and efficiency of poly(dT) translation has been revealed in cell-free systems from wild-type E coli and streptomycin--resistant mutants with altered ribosomal protein S12. Different factors promoting misreading of poly(U) such as aminoglycoside antibiotics and Mg2+ ions also stimulate poly(dT) translation. The effect of the antibiotics on poly(U) translation efficiency and misreading as well as on poly(dT) decoding is characterised by the same order: neomycin greater than kanamycin greater than streptomycin. S12 mutants ribosomes are less erroneous in poly(U) translation and less efficient in poly(dT) decoding. The data obtained are in good agreement with the hypothesis of stereospecific stabilization of codon-anticodon complexes by the ribosome decoding centre.

Anti-Bacterial Agents↗