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Z Földes-Papp

Publications and source records attributed to Z Földes-Papp.

At least 19 recordsLinked to original sources
PubMed2006

Chronic obstructive pulmonary disease and oxidative stress.

The respiratory tract as the main entrance for various inhalative substances has great potential to generate reactive species directly or indirectly in excess. Thus, heavy smokers are at high risk for development, impairment and failed response to treatment of chronic obstructive pulmonary disease (COPD). The article is an update regarding the influence of reactive oxygen (ROS) and nitrogen (RNS) species on COPD; however, we do not intend to describe ROS and RNS actions on the entire lung tissue. Here, we focus on the airways, because in human most of the described effects of ROS and RNS species are measured on respiratory epithelial cells obtained by bronchoscopy. ROS and RNS species are physiological compounds in cells and risk factors for several respiratory diseases. In general, both kinds of species are thermodynamically stabile, but their reaction behaviors in cellular environments are very different. For example, the life times of the superoxide anion radical range from micro/milliseconds up to minutes and even hours in in-vitro model systems. Oxidative stress by cigarette smoke was investigated in detail by the authors of this article. In addition, original studies by the authors on the amount of fine particulate matter and trace elements in lung biopsies after defined inhalation indicate a distortion of the equilibrium between oxidants and antioxidants. We also try to present some modern views with respect to genomic medicine for future therapeutic perspectives, although this is an upcoming sector of COPD therapy.

Animals↗
PubMed2001

Ultrasensitive detection and identification of fluorescent molecules by FCS: impact for immunobiology.

An experimental application of fluorescence correlation spectroscopy is presented for the detection and identification of fluorophores and auto-Abs in solution. The recording time is between 2 and 60 sec. Because the actual number of molecules in the unit volume (confocal detection volume of about 1 fl) is integer or zero, the fluorescence generated by the molecules is discontinuous when single-molecule sensitivity is achieved. We first show that the observable probability, N, to find a single fluorescent molecule in the very tiny space element of the unit volume is Poisson-distributed below a critical bulk concentration c*. The measured probability means we have traced, for example, 5 x 10(10) fluorophore molecules per ml of bulk solution. The probability is related to the average frequency, C, that the volume of detection contains a single fluorescent molecule and to the concentration, c, of the bulk solution. The analytical sensitivity of an assay is calculated from the average frequency C. In the Goodpasture experiment, we determined as analytical sensitivity a probability of 99.1% of identifying one single immune complex. Under these conditions, a single molecule event is proven. There exist no instrumental assumptions of our approach on which the experiment itself, the theoretical background, or the conclusion are based. Our results open up a broad field for analytics and diagnostics in solution, especially in immunology.

Anti-Glomerular Basement Membrane Disease↗
PubMed2001

Single molecule DNA sequencing in submicrometer channels: state of the art and future prospects.

We demonstrate a new method for single molecule DNA sequencing which is based upon detection and identification of single fluorescently labeled mononucleotide molecules degraded from DNA-strands in a cone shaped microcapillary with an inner diameter of 0.5 microm. The DNA was attached at an optical fiber via streptavidin/biotin binding and placed approximately 50 microm in front of the detection area inside of the microcapillary. The 5'-biotinylated 218-mer model DNA sequence used in the experiments contained 6 fluorescently labeled cytosine and uridine residues, respectively, at well defined positions. The negatively charged mononucleotide molecules were released by addition of exonuclease I and moved towards the detection area by electrokinetic forces. Adsorption of mononucleotide molecules onto the capillary walls as well as the electroosmotic (EOF) flow was prevented by the use of a 3% polyvinyl pyrrolidone (PVP) matrix containing 0.1% Tween 20. For efficient excitation of the labeled mononucleotide molecules a short-pulse diode laser emitting at 638 nm with a repetition rate of 57 MHz was applied. We report on experiments where single-stranded model DNA molecules each containing 6 fluorescently labeled dCTP and dUTP residues were attached at the tip of a fiber, transferred into the microcapillary and degraded by addition of exonuclease I solution. In one experiment, the exonucleolytic cleavage of 5-6 model DNA molecules was observed. 86 photon bursts were detected (43 Cy5-dCMP and 43 MR121-dUMP) during 400 s and identified due to the characteristic fluorescence decay time of the labels of 1.43+/-0.19 ns (Cy5-dCMP), and 2.35+/-0.29 ns (MR121-dUMP). The cleavage rate of exonuclease I on single-stranded labeled DNA molecules was determined to 3-24 Hz under the applied experimental conditions. In addition, the observed burst count rate (signals/s) indicates nonprocessive behavior of exonuclease I on single-stranded labeled DNA.

Base Sequence↗
PubMed2001

Fluorescently labeled model DNA sequences for exonucleolytic sequencing.

We describe here the enzyme-catalyzed, low-density labeling of DNAs with fluorescent dyes. Firstly, for "natural" template DNAs, dNTPs were partially substituted in the labeling reactions by the respective fluorophore-bearing analogs. The DNAs were labeled by PCR using Taq DNA polymerase. The covalent incorporation of dye-dNTPs decreased in the following order: rhodamine-green-5-dUTP (Molecular Probes, the Netherlands), tetramethylrhodamine-4-dUTP (FluoroRed, Amersham Pharmacia Biotech), Cy5-dCTP (Amersham Pharmacia Biotech). Exonucleolytic degradation by the 3'-->5' exonuclease activity of T7 DNA polymerase (wild type) in the presence of excess reduced thioredoxin proceeded to complete breakdown of the labeled DNAs. The catalytic cleavage constants determined by fluorescence correlation spectroscopy were between 0.5 and 1.5 s(-1) at 16 degrees C, normalized for the covalently incorporated dye-nucleotides. Secondly, rhodamine-green-X-dUTP (Roche Diagnostics), tetramethylrhodamine-6-dUTP (Roche Diagnostics), and Cy5-dCTP were covalently incorporated into the antisense strand of "synthetic" 218-b DNA template constructs (master sequences) at well defined positions, starting from the primer binding site, by total substitution for the naturally occurring dNTPs. The 218-b DNA constructs were labeled by PCR with a thermostable 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase which we have selected. The advantage of the special, synthetic DNA constructs as compared to natural DNAs lies in the possibility of obtaining tailor-made nucleic acids, optimized for testing the performance of exonucleolytic sequencing. The number of incorporated fluorescent nucleotides determined by complete exonucleolytic degradation and fluorescence correlation spectroscopy were six out of six possible incorporations for rhodamine-green-X-dUTP and tetramethylrhodamine-6-dUTP, respectively. Their covalent and base-specific incorporations were confirmed by the novel analysis methodology of re-sequencing (i.e. mobility-shift gel electrophoresis, reversion-PCR and re-sequencing) first developed in the paper Földes-Papp et al. (2001) and in this paper. This methodology was then used by other groups within the whole sequencing project.

Base Sequence↗
PubMed2001

Fluorescent high-density labeling of DNA: error-free substitution for a normal nucleotide.

The enzymatic incorporation of deoxyribonucleoside triphosphates by a thermostable, 3'-->5' exonuclease deficient mutant of the Tgo DNA polymerase was studied for PCR-based high-density labeling of 217-bp "natural" DNA in which fluorescent-dUTP was substituted completely for the normal dTTP. The amplified DNA carried two different sorts of tethered dye molecules. The rhodamine-green was used for internal tagging of the DNA. Since high-density incorporation of rhodamine-green-X-dUTP led to a substantial reduction (quenching) of the rhodamine-green fluorescence, a second "high" quantum yield label, Cy5, was inserted via a 5'-tagged primer in order to identify the two-color product. A theoretical concept of fluorescence auto- and cross-correlation spectroscopy developed here was applied to quantify the DNA sequence formed in terms of both the number of two-color fluorescent molecules and the number of covalently incorporated rhodamine-green-X-dUMP residues. The novel approach allowed to separate optically the specific DNA product. After complete, exonucleolytic degradation of the two-color DNA we determined 82-88 fluorescent U* labels incorporated covalently out of 92 maximum possible U* incorporations. The heavily green-labeled DNA was then isolated by preparative mobility-shift electrophoresis, re-amplified in a subsequent PCR with normal deoxyribonucleoside triphosphates, and re-sequenced. By means of this novel methodology for analyzing base-specific incorporations that was first developed here, we found that all fluorescent nucleotides and the normal nucleotides were incorporated at the correct positions. The determined labeling efficiency of 0.89-0.96 indicated that a fraction of the substrate analog was not bearing the fluorophore. The results were used to guide developments in single-molecule DNA sequencing. The labeling strategy (principal approach) for PCR-based high-density tagging of DNA, which included an appropriate thermostable DNA polymerase and a suitable fluorescent dye-dNTP, was developed here.

Base Sequence↗
PubMed2001

Towards a general procedure for sequencing single DNA molecules.

In this paper we report on the latest technical advances towards single molecule sequencing, a useful method currently developed especially for fast and easy de novo sequencing. Different approaches for complete labeling of DNA with fluorescent dyes are described. In addition, the experimental set-up for the sequencing process is shown. We demonstrate the ability to purify the buffer and enzyme solutions. Inorganic buffers were purified down to at least 20 fM of remaining fluorescent impurities. The exonuclease buffer solution could be cleaned down to 0.8 pM whereby its full activity was kept. Finally, we show a selection procedure for beads and present the data of a model experiment, in which immobilized DNA is degraded by an exonuclease within a polymethylmethacrylate (PMMA) microstructure. Furthermore, the mathematical processing of the obtained raw data is described. A first complete experimental cycle is shown, combining all preparatory steps which are necessary for single molecule sequencing in microstructures.

Biochemistry↗
PubMed2001

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen.

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.

Antigen-Antibody Complex↗
PubMed2001

Quantitative two-color fluorescence cross-correlation spectroscopy in the analysis of polymerase chain reaction.

We present results of an approach in which low-density labeled DNA itself provides an amplification of the cross-correlated fluorescent signal in the two-color cross-correlation function. Tetramethylrhodamine-4-dUTP and Cy5-dCTP are incorporated by polymerase chain reaction at multiple positions of the same 217 bp target DNA. We call this novel approach the 'two-color FCS signal amplification'. The signal amplification is an example for interactions of two ligands with different colors at multiple positions of the same target.

Carbocyanines↗
PubMed1999

The incipient stage in thrombin-induced fibrin polymerization detected by FCS at the single molecule level.

We used fluorescence correlation spectroscopy (FCS) to study the activation of fibrinogen by thrombin and the subsequent aggregation of fibrin monomers into fibrin polymers at a very low and at physiological fibrinogen concentrations. In the labeling procedure used the fibrinogen was randomly labeled and the label was bound to the fibrinopeptide A and/or to the part of fibrinogen which after activation takes part in fibrin formation. We measured a diffusion coefficient for fibrinogen of 2.48 x 10(-7) +/- 0.10 x 10(-7) cm2/s. After activation with thrombin both fibrinopeptide A and fibrin polymerization products could be demonstrated. From our findings we suggest a model for the formation of a three-dimensional network as two parallel processes, elongation and branching and that fibrin oligomers are not only intermediates in the polymerization process but also are substrates for branching.

Algorithms↗
PubMed1998

Fluorescence correlation spectroscopy of enzymatic DNA polymerization.

We show that fluorescence correlation spectroscopy (FCS) can be used as a reliable, simple, and fast tool for detecting products of the polymerase chain reaction (PCR). By use of autocorrelation experiments, it is demonstrated that fluorescent 217-bp DNA fragments can be detected at very low initial ss M13mp18(+) DNA and tetramethylrhodamine-4-dUTP concentrations and that these polymers are cleaved by the chosen restriction enzymes. A FCS calibration curve is presented, where the translational diffusion times of different size DNA fragments are plotted versus the number of base pairs they contain. At zero and very low template concentrations a large "background" species emerges, which is a reflection of the experimental conditions chosen and the extremely high sensitivity of FCS. The relative amount of nonspecific product formation is less than 1%. The ease by which a FCS measurement can be performed (a few minutes at most) also enables the technique to be used as an effective screening method.

Base Composition↗
PubMed1998

Fluorescence cross-correlation: a new concept for polymerase chain reaction.

In this article we present a new concept for the detection of any specifically amplified target DNA sequences in multiple polymerase chain reactions (PCR) based on fluorescence correlation spectroscopy (FCS). The accumulation of double-stranded target DNA is monitored by the cross-correlated fluorescence signals provided by two amplification primers which are 5'-tagged with two different kinds of fluorophores (Rhodamine-Green and Cy5). Only the amplified target DNA sequence carrying both primers is observed. Its signal emerges from the background of non-incorporated or non-specifically incorporated primers. Down to 10-25 initial copy numbers of the template in the PCR compartment DNA can presently be detected. No external or internal standards are required for determining the size and the amplified copy number of specific DNA. The PCR amplification process is started with all ingredients in a single compartment (e.g. of a microtiter plate), in which amplification and measurement are performed. This eliminates the need for post-PCR purification steps. The homogeneous one-tube approach does not depend on fluorescence energy transfer between the fluorogenic dyes. Thus, it does not interfere with the enzymatic amplification reaction of PCR and allows the continued use of different conditions for amplifying DNA. The results exemplified by PCR-amplified 217-bp and 389-bp target DNA sequences demonstrate that the analysis based on two-color fluorescence cross-correlation is a powerful method for simplifying the identification of targets in PCR for medical use. For this purpose, an instrument optimized for two-color excitation and detection of two-color emission has been developed, incorporating the principle of confocal arrangement.

Base Sequence↗
PubMed1996

Fractals for multicyclic synthesis conditions of biopolymers. Examples of oligonucleotide synthesis measured by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography.

We have developed models of patterns for nucleotide chain growth. These patterns are measurable by high-performance capillary electrophoresis and ion-exchange high-performance liquid chromatography in crude products of solid-phase synthesized 30mer and 65mer oligodeoxyribonucleotide target sequences N. We introduce mathematical methods for finding characteristic values d(o) and p(o) for constant chemical modes of growth as well as d and p for non-constant chemical modes of growth (d = probability of propagation, p = probability of termination). These methods are employed by presenting the accompanying computer software developed by us in C code, Mathematica R languages, and Fortran. Characteristic values of the parameters d, p, and the target nucleotide length N describe the complete composition of the crude product. From this we have developed the relation 2 - [N/(N - 1)]/Da, measurable(N,d) as a universal quantitative measure for multicyclic synthesis conditions (D, fractal dimension and similarity exponent, respectively). We use this mathematical treatment to compare the efficiency of oligodeoxyribonucleotide syntheses of different target length N on polymer support materials. Further, we analyze selected syntheses of short and long oligodeoxyribonucleotides as well as single-stranded DNA sequences by well-known empirical autocorrelation, fast Fourier transformation, and embedding dimension techniques.

Base Sequence↗
PubMed1995

Fractal dimension of error sequence dynamics in quantitative modeling of syntheses of short oligonucleotide and single-stranded DNA sequences.

Oligonucleotides are becoming more and more important in molecular biomedicine; for example, they are used as defined primers in polymerase chain reaction and as antisense oligonucleotides in gene therapy. In this paper, we model the dynamics of polymer-supported oligonucleotide synthesis to an inverse power law of driven multi-cycle synthesis on fixed starting sites. The mathematical model is employed by presenting the accompanying view of error sequences dynamics. This model is a practical one, and is applicable beyond oligonucleotide synthesis to dynamics of biological diversity. Computer simulations show that the polymer support synthesis of oligonucleotides and single-stranded DNA sequences in iterated cyclic format can be assumed as scale-invariant. This synthesis is quantitatively described by nonlinear equations. From these the fractal dimension Da (N,d) is derived as the growth term (N = number of target nucleotides, d = coupling probability function). Da(N,d) is directly measurable from oligonucleotide yields via high-performance liquid chromatography or capillary electrophoresis, and quantitative gel electrophoresis. Different oligonucleotide syntheses, including those with large-scale products can be directly compared with regard to error sequences dynamics. In addition, for short sequences the fractal dimension Da (N,d) is characteristic for the efficiency with which a polymer support of a given load allows oligonucleotide chain growth. We analyze the results of separations of crude oligonucleotide product from the synthesis of a 30 mer. Preliminary analysis of a 238 mer single-stranded DNA sequence is consistent with a simulated estimate of crude synthesis product, although the target sequence itself is not detectable. We characterize the oligonucleotide support syntheses by simulated and experimentally determined values of the fractal dimension Da (N,d0) within limitations (d0 = constant (average) coupling probability).

Animals↗
PubMed1992

The animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases--approaches to molecular arrangements of functional parts and oxidative changes.

The molecular structures of animal and human plasma membrane (Ca(2+)+Mg2+)-ATPases are not completely understood in part due to the fact that no suitable single crystal is available. The elucidation of the two-dimensional structure is in progress. The amino acid sequences of human erythrocyte and rat plasma membrane Ca2+ pump isoforms as well as of the pig smooth muscle plasma membrane Ca2+ pump are already known. This article reviews the present state of the knowledge in (Ca(2+)+Mg2+)-ATPase research of animal and human plasma membranes performed in the past few years, concerning in particular arrangements of proteolytically cleaved fragments, and relations between the erythrocyte (Ca(2+)+Mg2+)-ATPase in situ and the purified red cell enzyme, oxidative changes. Results of different experimental approaches concerning the structure of (Ca(2+)+Mg2+)-ATPases rather than the applications of the methods used are emphasized.

Animals↗
PubMed1986

Molecular characterization of the in situ red cell membrane calcium pump by limited proteolysis.

In inside-out red cell membrane vesicles active calcium transport and the formation of the enzyme-phosphate complex (EP) of the calcium pump were simultaneously investigated and the effects of a limited proteolytic digestion examined. In order to visualize the proteolyzed EP forms we have induced the formation of a maximum level EP from [gamma-32P]ATP in the presence of Ca2+ + La3+ and applied a good-resolution acidic discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Proteolysis of inside-out vesicle membranes by trypsin, Pronase, papain, or chymotrypsin produces a calmodulin-like activation of the calcium pump, abolishes its calmodulin sensitivity, and decreases the original 140-kDa EP complex to a limit polypeptide of 80 kDa. Trypsin digestion produces another major intermediary fragment of 90 kDa, which is still a low-activity calmodulin-sensitive form of the pump. The red cell calcium pump is activated by trypsin both in the absence and presence of Ca2+ during digestion although the rate of activation and the appearance of the 80-kDa polypeptide are enhanced by Ca2+. If proteolytic digestion is carried out by chymotrypsin, a calmodulin-insensitive maximum activation of the calcium pump coincides with the formation of a 125-130-kDa EP-forming polypeptide. Chymotrypsin and carboxypeptidase A have synergistic effects on the formation of this latter high-activity species. Based on these data we suggest a probable molecular arrangement for the functional parts of the red cell membrane calcium pump.

Adenosine Triphosphatases↗
PubMed1986

Demonstration of two distinct calcium pumps in human platelet membrane vesicles.

Membrane vesicles from human platelets were prepared by various disruption and isolation techniques reported in the literature to yield fractions of predominantly surface or intracellular membrane origin. ATP + Mg2+-dependent Ca2+ accumulation and the formation of acylphosphate intermediates of the calcium pump(s) were followed in parallel experiments, and the consequences of a limited proteolysis of the membranes examined. In all types of preparations active Ca2+ uptake had both oxalate-sensitive and insensitive fractions and calmodulin had no effect on the rate of Ca2+ uptake. Limited proteolysis by trypsin eliminated oxalate-sensitive Ca2+ uptake while it had no effect on the oxalate-insensitive fraction. The Ca2+-induced EP complex had an apparent molecular mass of 100-110 kDa in all of the preparations, the EP showing a broad or even duplicated line in most autoradiographies. Mild trypsin digestion resulted in the formation of 80-, 55-, and 35-kDa phosphorylated fragments. The 80-kDa fragment corresponded to the limit polypeptide found in the proteolyzed erythrocyte membrane Ca2+ pump, its phosphorylation was stimulated by lanthanum, and it appeared in a different time course than the smaller fragments. The molecular mass and the formation pattern of the latter species corresponded to the tryptic fragments in the sarcoplasmic reticulum Ca2+ pump. Based on these results we suggest that platelet membrane preparations contain two types of Ca2+ pump proteins, one similar to the sarcoplasmic reticulum-type and the other to the erythrocyte-type enzyme.

Adenosine Triphosphatases↗
PubMed1984

Enzymatic t-butyl hydroperoxide reduction on human erythrocyte membranes--NADPH and GSH dependent activities.

A NADPH-dependent t-butyl hydroperoxide ( TBH )-reducing activity independent of glutathione was found in addition to glutathione peroxidase activity bound to erythrocyte membranes. In "hypotonic" and "isotonic" membranes the NADPH-dependent TBH -reducing activity amounted to about 0.34 mu kat /l red blood cells (RBC) and the glutathione peroxidase activity to 0.32 mu kat /l RBC. The activities do not appear to be additive. The membrane association of the enzymes is independent of ionic strength. Under hypotonic condition about 0.2% of the total cellular catalase activity were bound to the membrane but none in "isotonic" membranes. The bound catalase appears to exhibit a glutathione dependent peroxidase activity. Membrane-bound haemoglobin exhibited a quasi- TBH -reductase activity which was inhibited by azide and cyanide.

Catalase↗