Biomedical subjects

F Jelen

Publications and source records attributed to F Jelen.

8 recordsLinked to original sources

PubMed2003

Canonical protein inhibitors of serine proteases.

Serine proteases and their natural protein inhibitors are among the most intensively studied protein complexes. About 20 structurally diverse inhibitor families have been identified, comprising alpha-helical, beta sheet, and alpha/beta proteins, and different folds of small disulfide-rich proteins. Three different types of inhibitors can be distinguished based on their mechanism of action: canonical (standard mechanism) and non-canonical inhibitors, and serpins. The canonical inhibitors bind to the enzyme through an exposed convex binding loop, which is complementary to the active site of the enzyme. The mechanism of inhibition in this group is always very similar and resembles that of an ideal substrate. The non-canonical inhibitors interact through their N-terminal segment. There are also extensive secondary interactions outside the active site, contributing significantly to the strength, speed, and specificity of recognition. Serpins, similarly to the canonical inhibitors, interact with their target proteases in a substrate-like manner; however, cleavage of a single peptide bond in the binding loop leads to dramatic structural changes.

Binding Sites↗

PubMed2002

New approaches in the development of DNA sensors: hybridization and electrochemical detection of DNA and RNA at two different surfaces.

Up to now, the development of the electrochemical DNA hybridization sensors relied on solid electrodes, on which both the hybridization and detection steps have been performed. Here we propose a new method in which the DNA hybridization is performed at commercially available magnetic beads and electrochemical detection on detection electrodes (DE). Due to minimum nonspecific DNA adsorption at the magnetic beads, very high specificity of the DNA hybridization is achieved. Optimum DE can be chosen only with respect to the given electrode process. It is shown that high sensitivity and specificity in the detection of relatively long target DNAs can be obtained (a) by using cathodic stripping voltammetry at mercury or solid mercury amalgam DEs for the determination of purine bases, released from DNA by acid treatment, and (b) by enzyme-linked immunoassay of target DNA modified by osmium tetroxide,2,2'-bipyridine (Os,bipy) at carbon DEs. Direct determination of Os,bipy at mercury and carbon electrodes is also possible.

Biosensing Techniques↗

PubMed1997

Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions.

Adsorption↗

PubMed1991

Osmium tetroxide reactivity of DNA bases in nucleotide sequencing and probing of DNA structure.

Osmium tetroxide, 2,2'-bipyridine (Os,bipy) has been widely applied as a probe of the DNA structure. To obtain information about reactivity of DNA bases toward this probe synthetic homopolynucleotides poly(dT), poly(dC), poly(dG) and poly(dA) were treated with Os,bipy and the content of modified bases measured by stripping voltammetry and absorption spectrophotometry. After 20 hours' treatment strong modification of poly(dT) and poly(dC) and weak modification of poly(dG) were observed, while no modification was detected in poly(dA). At short incubation times under conditions close to those usually used in probing the DNA structure the extent of poly(dT) modification was more than 10 times higher than that of poly(dC). Thus, in single-stranded DNA Os,bipy reacts with T much greater than C and G. Due to the fast reaction of thymines with Os,bipy (and osmium tetroxide, pyridine) these chemicals can be applied in Maxam-Gilbert nucleotide sequencing as agents specific for thymines in single-stranded DNA.

2,2'-Dipyridyl↗

PubMed1986

Chemically reversible electroreduction of guanine in a polynucleotide chain.

It was shown that synthetic polynucleotides containing guanine display in cyclic voltammetry (CV) an anodic peak close to -0.3 V (against a saturated calomel electrode). A condition for the appearance of this peak is the previous polarization of the mercury electrode to sufficiently negative potentials (around -1.8 V). The results of CV measurements with electrode polarization by repeated cycles indicate that in negative potentials there is a reduction of guanine residues and in the anodic process reoxidation of the reduction product to guanine. This chemically reversible process takes place even when a polynucleotide contains adenine and/or cytosine residues in addition to guanine, where reduction leads to the formation of products blocking the electrode surface.

DNA↗

PubMed1986

Cyclic voltammetry of DNA at a mercury electrode: an anodic peak specific for guanine.

Synthetic homopolyribonucleotides poly(A), poly(U), poly(C), and poly(G), poly(A, G, U), apurinic acid and native and denatured DNA from calf thymus were analyzed by means of cyclic voltammetry (CV) using a hanging mercury drop electrode. It was shown that guanine containing polynucleotides, i.e. poly(G), poly(A, G, U) and DNA yield an anodic peak of guanine in the vicinity of a potential of -0.3 V (against a saturated calomel electrode). The guanine peak appeared only at a sufficiently negative switching potential (about -2 V). The appearance of the guanine peak was conditioned by a reduction of guanine residues in the region of the switching potential and reoxidation of the reduction product in the vicinity of -0.3 V. Native and thermally denatured DNAs were investigated under the conditions of both complete and incomplete coverage of the electrode in various background electrolytes. Both DNA forms yielded anodic CV peaks of guanine with the peak of denatured DNA being always higher than that of native DNA. Irradiation of native DNA with relatively small doses of gamma radiation (5-120 Gy) resulted in an increase of the anodic peak. A comparison of changes induced by gamma radiation in the anodic (guanine) and cathodic (reduction of adenine and cytosine) peaks showed a steeper increase of the cathodic peak as compared to that of the anodic one. It has been concluded that in the given dose range the DNA double-helical structure is mainly damaged in the adenine-thymine rich regions.

Apurinic Acid↗

PubMed1985

Nucleotide sequence-dependent opening of double-stranded DNA at an electrically charged surface.

It has been shown earlier that the DNA double helix is opened due to a prolonged contact of the DNA molecule with the surface of the mercury electrode. At neutral pH, the opening process is relatively slow (around 100 s), and it is limited to potentials close to -1.2 V (against SCE). The opening of the double helix has been explained by strains in the DNA molecule due to strong repulsion of the negatively charged phosphate residues from the electrode surface where the polynucleotide chain is anchored via hydrophobic bases. Interaction of the synthetic ds polynucleotides with alternating nucleotide sequences/poly(dA-dT).poly (dA-dT), poly (dA-dU).poly (dA-dU), poly (dG-dC).poly (dG-dC)/ and homopolymer pairs/poly (dA).poly (dT), poly (rA).poly (rU) and poly (dG).poly (dC)/ with the hanging mercury drop electrode has been studied. Changes in reducibility of the polynucleotides were exploited to indicate opening of the double helix. A marked difference in the behaviour was observed between polynucleotides with alternating nucleotide sequence and homopolymer pairs: opening of the double-helical structures of the former polynucleotides occurs at a very narrow potential range (less than 100 mV) (region U), while with the homopolymer pairs containing A X T or A X U pairs, the width of this region is comparable to that of natural DNA (greater than 200 mV). In contrast to natural DNA, the region U of homopolymer pairs is composed of two distinct phases. No region U was observed with poly (dG).poly (dC). In polynucleotides with alternating nucleotide sequence, the rate of opening of the double helix is strongly dependent on the electrode potential in region U, while in homopolymer pairs, this rate is less potential-dependent. It has been assumed that the difference in the behaviour between homopolymer pairs and polynucleotides with alternating nucleotide sequence is due to differences in absorbability of the two polynucleotide chains in the molecule of a homopolymer pair (resulting from different absorbability of purine and pyrimidine bases) in contrast to equal adsorbability of both chains in a polynucleotide molecule with alternating nucleotide sequence. It has been shown that the mercury electrode is a good model of biological surfaces (e.g. membranes), and that the nucleotide sequence-dependent opening (unwinding) of the DNA double helix at electrically charged surfaces may play an important role in many biological processes.

Adsorption↗

PubMed1984

Osmium-induced alteration in DNA structure.

In the presence of pyridine and other ligands osmium tetroxide binds covalently to pyrimidine bases in DNA. Properties of osmium-modified native and denatured calf thymus DNA, and plasmid Co1E1 DNA were investigated by means of differential pulse polarography, absorption spectrophotometry, circular dichroism, agarose gel electrophoresis, and nuclease S1 digestion. A great difference in the reaction kinetics of native and denatured DNAs with osmium, pyridine was observed. On the ground of the slow stepwise reaction kinetics of native DNA in the initial stage of its modification by osmium it has been suggested that the primary reaction sites do not include bases contained in the intact double helix. Osmium binding to sporadic primary reaction sites (represented e.g. by bases in the vicinity of a single-strand break) in native calf thymus DNA resulted in local changes in DNA conformation limited to a close neighbourhood of the binding site. At higher osmium/nucleotide ratios disordering of the DNA structure over a region extending beyond the immediate binding site was observed. With denatured DNA the same type of structure disordering was detected already in the initial stage of the reaction at osmium/nucleotide ratios as low as 0.01. Osmium binding to the supercoiled Co1E1 DNA resulted in its relaxation without nicking and it increased its sensitivity to linearization by cleavage with nuclease S1. The behaviour of Co1E1 DNA has been explained by the formation of a denatured region in the molecule (accompanied by a coupled loss of duplex and superhelical turns). It has been suggested that osmium can be used to label and to visualize distorted regions in the DNA double helix.

Animals↗