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Biomedical subjects

L Wieczorek

Publications and source records attributed to L Wieczorek.

At least 19 recordsLinked to original sources
PubMed2001

Solution- and solid-phase synthesis of components for tethered bilayer membranes.

The synthesis of the novel compound PhCH(2)SS(C(24)H(44)N(4)O(10))(C(20)H(41)) (5) for the preparation of tethered bilayer membranes is described. The compound is the all-amide analogue of the previously reported ester-containing membrane-forming material PhCH(2)SS(C(24)H(40)O(14))(C(20)H(41)) (1). The advanced intermediate (C(20)H(41)) C(16)H(28)N(3)O(8) (17) was prepared from the same starting materials using both solution-phase (13% yield) and solid-phase (81% yield) techniques. Monolayers on gold derived from 5 have been analyzed by ellipsometry and FTIR. The monolayers exhibit thicknesses similar to monolayers derived from 1 and possess H-bonded amide functionality.

Journal Article↗
PubMed2001

Tethered-bilayer lipid membranes as a support for membrane-active peptides.

An immunosensing device, comprising a lipid membrane incorporating ion channels tethered to the surface of a gold electrode, has been reported [Cornell, Braach-Maksvytis, King, Osman, Raguse, Wieczorek and Pace (1997) Nature (London) 387, 580-583]. The present article describes key steps in the assembly of the device and provides further evidence for its proposed sensing mechanism.

Computer Simulation↗
PubMed1999

The gramicidin-based biosensor: a functioning nano-machine.

Biosensors combine a biological recognition mechanism with a physical transduction technique. In nature, the transduction mechanism for high sensitivity molecular detection is the modulation of the cell membrane ionic conductivity through specific ligand-receptor binding-induced switching of ion channels. This effects an inherent signal amplification of six to eight orders of magnitude, corresponding to the total ion flow arising from the single channel gating event. Here we describe the first reduction of this principle to a practical sensing device, which is a planar impedance element composed of a macroscopically supported synthetic bilayer membrane incorporating gramicidin ion channels. The membrane and an ionic reservoir are covalently attached to an evaporated gold surface. The channels have specific receptor groups attached (usually antibodies) that permit switching of gramicidin channels by analyte binding to the receptors. The device may then be made specific for the detection of a wide range of analytes, including proteins, drugs, hormones, antibodies, DNA, etc., currently in the 10(-7)-10(-13) M range. It also lends itself readily to microelectronic fabrication and signal transduction. By adjusting the surface density of the receptors/channel components during fabrication, the optimum sensitivity range of the device may be tuned over several orders of magnitude.

Anti-Bacterial Agents↗
PubMed1999

Erratum

The paper 'Interference of rheumatoid factor activity by aspartame, a dipeptide methyl ester' by Paul A. Ramsland, Bahereh F. Movafagh, Morris Reichlin and Allen B. Edmundson, J. Mol. Recognit. 1999; 12: 249--257, was published without the required colour plates. The publisher would like to apologise for this omission. The article is reprinted here in full. Please replace the previously published pages with those following. The electronic version of the article, including the colour plates, can be downloaded from the Wiley Interscience website at http://www.interscience.wiley.com. The plates have been included in the original paper, which appeared in Issue 4 of the journal.

Journal Article↗
PubMed1998

Kinetics of the competitive response of receptors immobilised to ion-channels which have been incorporated into a tethered bilayer.

A competitive ion channel switch (ICS) biosensor has been modelled yielding ligand mediated monomer-dimer reaction kinetics of gramicidin (gA) ion-channels within a tethered bilayer lipid membrane. Through employing gramicidin A, functionalized with the water-soluble hapten digoxigenin, it is possible to cross-link gramicidin to antibody fragments tethered at the membrane/aqueous interface. The change in ionic conductivity of the channel dimers may then be used to measure the binding kinetics of hapten-protein interactions at the membrane surface. The approach involves measuring the time dependence of the increase in impedance following the addition of a biotinylated antibody fragment (b-Fab'), which cross-links the functionalized gramicidin monomers in the outer layer of the lipid bilayer to tethered membrane spanning lipid. The subsequent addition of the small molecule digoxin, (M(r) 781 Da), competes with and reverses this interaction. The model provides a quantitative description of the response to both the cross-linking following the addition of the b-Fab' and the competitive displacement of the hapten by a water-soluble small analyte. Good agreement is obtained with independent measures of the cross-linking reaction rates of the gramicidin monomer-dimer and the b-Fab: hapten complex. The rate and amplitude of the competitive response is dependent on concentration and provides a fast and sensitive detection technique. Estimates are made of the concentration of gramicidin monomers in both the inner and outer monolayer leaflets of the membrane. This is used in the calculation of the gramicidin monomer/dimer equilibrium constant, K2D3. Other considerations include the membrane impedance limit set by the membrane leakage which is also a function of the concentration of the gA monomer concentration, and the two-dimensional kinetic association constant k2D2, of the hapten: b-Fab' complex. The gA dimer concentration is dependent on both the concentration of gA-dig and of the tethered streptavidin: b-Fab' complexes. The model shows that the 2D dissociation constant k2D3(-1), must be at least 10 times faster than the 3D dissociation constant k3D2(-1) for digoxin to completely reverse the cross-linked hapten-receptor interaction at the membrane interface.

Biosensing Techniques↗
PubMed1997

A biosensor that uses ion-channel switches.

Biosensors are molecular sensors that combine a biological recognition mechanism with a physical transduction technique. They provide a new class of inexpensive, portable instrument that permit sophisticated analytical measurements to be undertaken rapidly at decentralized locations. However, the adoption of biosensors for practical applications other than the measurement of blood glucose is currently limited by the expense, insensitivity and inflexibility of the available transduction methods. Here we describe the development of a biosensing technique in which the conductance of a population of molecular ion channels is switched by the recognition event. The approach mimics biological sensory functions and can be used with most types of receptor, including antibodies and nucleotides. The technique is very flexible and even in its simplest form it is sensitive to picomolar concentrations of proteins. The sensor is essentially an impedance element whose dimensions can readily be reduced to become an integral component of a microelectronic circuit. It may be used in a wide range of applications and in complex media, including blood. These uses might include cell typing, the detection of large proteins, viruses, antibodies, DNA, electrolytes, drugs, pesticides and other low-molecular-weight compounds.

Biosensing Techniques↗
PubMed1993

Adverse events of subcutaneous recombinant human erythropoietin therapy: results of a controlled multicenter European study.

In a controlled European multicenter study, clinical tolerance of subcutaneously administered recombinant human erythropoietin (rh-EPO) therapy and its influence on the course of illness in 362 hemodialyzed patients (162 males, 200 females) from 16 European dialysis centers was studied. Of these, 181 patients served as a control group in the first year and received rh-EPO therapy in the second year. Of the 837 adverse events that occurred, 277 were classified as serious and 560 as nonserious. Thirty-two deaths have been reported for the study population: 18 in the control group and 14 in the therapy group. The individual analysis of the serious adverse events including death demonstrates a protective effect of rh-EPO on the high-risk cardiovascular situation of dialysis patients. Hypertension was no problem, and under rh-EPO therapy an increase in resistance to infection was observed. Subcutaneous rh-EPO treatment might have an even better safety profile than intravenous application.

Adult↗
PubMed1992

Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.

Parts of the large phosphorylated tegument protein, pp150, of human cytomegalovirus (HCMV) were expressed in bacteria. The resulting fusion proteins were tested in a Western blot (immunoblot) assay for reactivity with a monoclonal antibody against pp150, with a polyspecific rabbit antiserum, and with human reconvalescent-phase sera. Those fusion proteins that performed well in the Western blot assay were used as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against HCMV. Five different recombinant beta-galactosidase fusion proteins were evaluated by ELISA using 62 seropositive and 38 seronegative human serum samples. Of all the proteins tested, one peptide representing 162 amino acids of pp150 was superior to the others with regard to sensitivity and specificity. All sera known to be positive for antibodies against HCMV were identified by combining the results of the ELISAs with the different pp150 fusion proteins. Therefore, it appears that peptides from a single protein of HCMV might be sufficient to identify HCMV-seropositive individuals by recombinant ELISA.

Animals↗
PubMed1990

New enzyme-linked immunosorbent assay methods for measurement of serum erythropoietin levels and erythropoietin antibodies.

For clinical studies with erythropoietin (EPO), enzyme-linked immunosorbent assays for the determination of EPO and EPO antibodies were developed. Using polyclonal and monoclonal EPO antibodies in a sandwich technique, serum EPO levels greater than 10 pg/ml (corresponding to 1 mU/ml, calibrated with the 2nd WHO IRP EPO) can be determined. In 103 healthy blood donors, a mean (+/- SD) value of 36 +/- 19 pg EPO/ml was found. Very high EPO concentrations were found in patients suffering from myelodysplastic syndrome and aplastic anemia; elevated levels were associated with rheumatoid arthritis and myelomatosis. No EPO antibodies were detectable in EPO-treated patients.

Antibodies↗
PubMed1990

In vitro studies of erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes.

Erythropoietin-dependent regulation of erythropoiesis in myelodysplastic syndromes (MDS) was evaluated by measuring the in vitro response of primitive (BFU-E) and relatively mature (CFU-E) erythroid progenitors from 12 patients and from eight healthy donors to recombinant human erythropoietin (rhEPO), and by quantifying relationships between circulating EPO levels and progenitor cell frequencies in MDS marrow. Half-maximal growth of MDS CFU-E and BFU-E was detected at a 4-fold higher rhEPO concentration than required by control erythroid progenitors. Nine of the patients evaluated exhibited maximal growth of erythroid colonies at 5- to 20-fold higher than control saturating rhEPO concentrations. Circulating EPO levels in MDS patients were elevated, with a mean value approximately 35-fold higher than that of controls. The frequency of MDS marrow CFU-E and BFU-E was 57 +/- 42% and 18 +/- 9% of the mean control values, respectively. Correlation analysis of the relationships between MDS EPO levels and erythroid progenitors indicated that the anemia in MDS is not attributable to an abnormality in the capacity of EPO to induce the generation of CFU-E, but may be influenced by the BFU-E population, whose severe deficiency results in insufficient influx of EPO-responsive cells. Our findings therefore suggest that treatment of MDS patients with rhEPO may be of limited benefit, since the generation of BFU-E from more primitive ancestors and the initial growth requirements of these cells are not under the regulatory influence of this hormone.

Adolescent↗
PubMed1988

Evaluation of the stability of human erythropoietin in samples for radioimmunoassay.

Radioimmunoassays for erythropoietin are limited so far to a few specialized laboratories and this requires transport and storage of samples. We therefore tested the stability of immunoreactive erythropoietin in serum and plasma samples obtained from a uremic and a nonuremic anemic patient. No significant change in the concentration of immunoreactive erythropoietin was found in either serum or plasma samples for up to 14 days of storage. This type of stability was observed no matter whether the samples were stored at room temperature, 4 degrees C, or -20 degrees C. There was no difference between the estimates of erythropoietin in serum and heparinized plasma. Validity of the radioimmunoassay used in this study was demonstrated by parallelism of dilution curves of test specimens and the 2nd International Reference Preparation for erythropoietin and by a close correlation between the immunoreactivity and the bioactivity of the hormone, as assessed in the same samples by the exhypoxic polycythemic mouse bioassay. In conclusion the data obtained clearly indicate that the necessity of storage and transport of clinical samples does not limit the practicability of the radioimmunoassay for erythropoietin.

Anemia, Aplastic↗