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Biomedical subjects

G Löber

Publications and source records attributed to G Löber.

At least 19 recordsLinked to original sources
PubMed1999

Detection of the DeltaF508 mutation in the CFTR gene by means of time-resolved fluorescence methods.

A rapid recognition in the base sequence of nucleic acids is an important prerequisite toward the diagnosis of genetic diseases and their carrier states. We have developed a hybridisation method in which a fluorescently labeled oligonucleotide is used to detect point mutations in a target by a simple fluorescence lifetime analysis of the emission of the fluorescent label. We applied this method to detect the deltaF508 mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene in a model system and with biologically derived PCR product and discuss the potential generality of this method.

Base Sequence↗
PubMed1999

[Effect of hyperphosphorylation on structure of TAU protein].

Effect of hyperphosphorylation on structural properties and conformational stability of tau-protein was investigated by methods of circular dichroism and fluorescence decay. The normal protein displayed an unusual secondary structural elements--an extended left-handed helix. It is suggested that the structure of the normal tau-protein includes a globular C-terminal part with rigid extended tail, i.e., it is of a "tadpole" type. The normal protein structure is practically unaffected by changes in pH values. Hyperphosphorylation leads to some perturbation within the extended part of the protein molecule. Decrease in pH transforms the globular part of the hyperphosphorylated protein into the molten globule-like conformation.

Amino Acid Sequence↗
PubMed1998

Self-association of 8-anilino-1-naphthalene-sulfonate molecules: spectroscopic characterization and application to the investigation of protein folding.

It was suggested long ago that the reason for the considerable increase of 8-anilino-1-naphthalene-sulfonate (8-ANS) fluorescence intensity upon the transition from aqueous to organic solvents is the dissociation of 8-ANS associates. To clarify this point the dependence of spectral properties of the dye on concentration and solvent composition was investigated by means of steady-state and time-resolved fluorescence spectroscopy. It was shown that the increase of 8-ANS concentration leads to the changes in the shape of absorption and fluorescence spectra of the dye, accompanied by the decrease in its fluorescence decay time values. Such changes were observed in aqueous and organic solvents for Mg2+- and NH+4-salts of 8-anilino-1-naphthalene-sulfonateic acid and reflect the existence of self-association of the dye molecules in both media. However, the decrease in fluorescence intensity induced by the self-association of the probe molecules is too small to explain weak fluorescence of 8-ANS in water. At the same time, it expounds the difference between the decay times of protein-embedded 8-ANS molecules upon interaction of this probe with native and molten globule proteins.

Anilino Naphthalenesulfonates↗
PubMed1998

Variations of DNA damage in human lymphocytes after enflurane exposure in vitro.

The rate of DNA single strand-breaks in lymphocytes of 160 human donors were determined after enflurane (CAS 13838-16-9) exposure in vitro. The rate of DNA damage increased in relation to the exposed enflurane concentration. However, not every lymphocyte sample showed an increased rate of DNA damage; some samples showed an increased rate even after exposure to only 0.4 vol% enflurane and other samples showed no increased rate even after exposure to 4.0 vol% enflurane. After exposure to 0.6 vol% enflurane and more the increased rate of DNA damage is statistically significant. The DNA damage in the lymphocytes differs individually. The reasons may be genetic differences in DNA repair. In patients with DNA repair deficiencies anesthesia with enflurane may induce irreversible DNA damage.

Adolescent↗
PubMed1998

Molecular modelling and footprinting studies of DNA minor groove binders: bisquaternary ammonium heterocyclic compounds.

We report new quantitative footprinting data which reveal differences in binding constants of bisquaternary ammonium heterocyclic compounds (BQA) with AT-rich DNA sites depending on the ligand structure and on the size and sequence of the DNA binding site. In an attempt to understand the dependence of binding affinity on the ligand structure we have performed quantum-chemical AM1 calculations on the BQA compounds and on subunits to explore the conformational space and to calculate the electronic and structural features of individual ligand conformations. Due to the properties of the rotatable backbone bonds, there is a large number of possible conformations with almost equal energy. We present a new method for the calculation of the radius of curvature of molecular structures. Assuming that strong binders should have a shape complementary to the DNA minor groove, this measure is used to select the optimum conformations for DNA-drug binding. The approach yields the correct ligand conformation for SN6999, for which an X-ray DNA-drug structure is known. The curvature of the optimum conformations of all ligands is compared with the experimental binding constants. A correlation is found between curvature and binding constant provided other structural factors do not vary. Therefore, we conclude that within structurally similar BQA compounds the extent of curvature is the relevant quantity which modulates the binding affinity.

DNA↗
PubMed1997

Fluorescence and binding properties of phenazine derivatives in complexes with polynucleotides of various base compositions and secondary structures.

The interactions of two phenazine derivatives, one with a neutral chromophore (glycoside) and the other with a cationic one (quaternary salt), with various synthetic single- and double-stranded polynucleotides and natural DNA were studied by fluorescence techniques, conducting measurements of steady-state fluorescence intensity and polarization degree as well as fluorescence lifetime. These dyes show fluorescence quenching upon intercalation into the GC sequences of the double-stranded nucleic acids and an increase in fluorescence emission and lifetime upon incorporation into the AT and AU sequences. GC base pairs in continuous deoxynucleotide sequences were found to be preferred as binding sites for both phenazines, in contrast to AT base pairs. On the contrary, the continuous ribonucleotide GC sequence binds the phenazines more weakly than does the AU sequence. With regard to the interaction of the phenazines with single-stranded polynucleotides, a stacking interaction of the dye chromophores with the nucleic bases was observed. In that case the guanine residue quenches the cationic phenazine fluorescence, while the stacking interaction with the other bases results in an increase in the fluorescence quantum yield. Unlike the cationic dye, the fluorescence of the neutral phenazine was quenched by both purine bases.

Journal Article↗
PubMed1997

Conformational transitions provoked by organic solvents in beta-lactoglobulin: can a molten globule like intermediate be induced by the decrease in dielectric constant?

BACKGROUND: It is known that nonnative states of protein molecules can exist in living cells and can be involved in a number of physiological processes. It has also been established that the membrane surface can be responsible for the partial denaturation of proteins due to negative charges on it. The local decrease in the effective dielectric constant of water near the organic surface has been suggested to be an additional driving force for protein denaturation in the membrane field, but data to confirm this suggestion were lacking. RESULTS: Conformational transitions induced in beta-lactoglobulin by methanol, ethanol, isopropanol, dimethylformamide and dioxane were studied by near and far UV circular dichroism, steady-state tryptophan fluorescence and fluorescence decay of 8-anilinonaphthalene-1-sulfonate (8-ANS). The existence of at least two noncoinciding cooperative transitions has been established in all solvent systems studied. The first of these transitions describes the disruption of rigid tertiary structure in protein molecules, while the second reflects the formation of an expanded helical conformation typical of proteins in concentrated organic solvents. This means that the organic solvents provoke the formation of a denatured intermediate state with pronounced secondary structure and native-like compactness. We show that the positions of maxima in fI versus dielectric constant dependence virtually coincide for all five solvent systems studied. CONCLUSIONS: The decrease in the dielectric constant of the solvent induces in beta-lactoglobulin an equilibrium intermediate state. This state, being denatured, is relatively compact and has pronounced secondary structure and high affinity for the hydrophobic fluorescent probe 8-ANS, i.e. possesses all the properties of the molten globule intermediate state.

Anilino Naphthalenesulfonates↗
PubMed1997

Sequence-specific interactions of minor groove binders with the 154 base pair HindIII-RsaI restriction fragment of cDNA of the human Tau 40 protein involved in pathology of Alzheimer's disease.

The DNA minor groove binders netropsin, distamycin and four structurally related bisquaternary ammonium heterocycles (BQA), SN 6999, SN 6570, SN 6132 and SN 6131, were investigated for sequence-specific interactions with the 154 base pair fragment of cDNA of the human Tau 40 protein (h Tau 40 protein), involved in pathology of Alzheimer's disease. The base sequences 5' AATCTT 3', 5' AATATT 3' and 5' TTTCAATCTTTTTATTT 3' were identified as ligand specific binding sites and demonstrate the obvious dA.dT binding preference. Footprinting titration experiments were performed to estimate sequence-specific binding constants (KA). The KA-values were in the order of 10(6)M-1 and dependent on DNA base sequence as well as ligands used. The highest values estimated were for netropsin (KA = 5.0 x 10(6)M-1) and the quinoline derivative SN 6999 (KA = 6.2 x 10(6)M-1) binding to the sequence 5' ATAAT 3'. Microscopic binding constants are determined by the base sequence rather than by the length of dA.dT stretches. In the extended dA.dT run, 5' TTTCAATCTTTTTATTT 3', netropsin and distamycin binding tolerates the presence of two dG.dC base pairs, as indicated by nearly unaffected footprints. In contrast, the failure of BQAs to form footprints demonstrates their significantly decreased binding selectivity.

Alzheimer Disease↗
PubMed1997

Detection of DNA damage in stimulated human lymphocytes after adding cytostatic drugs in vitro. A model to demonstrate individual damage rates.

The DNA damaging effect of different concentrations of methotrexate, 6-mercaptopurine, 6-thioguanine and cisplatin was tested by nucleoid sedimentation in pokeweed mitogen (PWM)-stimulated lymphocytes of 16 healthy persons in vitro. The examined persons show an individual variation of DNA damage demonstrating individual differences in DNA repair. The method can be used to identify persons with a low DNA repair capacity, and possible cytogenetic side effects of cytostatic drugs can be calculated before starting a cancer therapy. In clinical practice the use of cytostatic drugs is limited because of the side effects on normal tissues. The cancer therapist can improve a cytostatic therapy when he obtains information about possible DNA damage of cytostatic drugs in cells of the patient at the beginning of the therapy.

Adult↗
PubMed1996

Use of fluorescence decay times of 8-ANS-protein complexes to study the conformational transitions in proteins which unfold through the molten globule state.

The conformational transitions starting with the native protein, passing the molten globule state and finally approaching the unfolded state of proteins was investigated for bovine carbonic anhydrase B (BCAB) and human alpha-lactalbumin (alpha-HLA) by means of fluorescence decay time measurements of the dye 8-anilinonaphthalene-1-sulphonic acid (8-ANS). Stepwise denaturation was realized by using the denaturant guanidinium chloride (GdmCl). It was shown that 8-ANS bound with protein yields a double-exponential fluorescence decay, where both decay times considerably exceed the decay time of free 8-ANS in water. This finding reflects the hydrophobic environment of the dye molecules attached to the proteins. The fluorescence lifetime of the short-time component is affected by protein association and can be effectively quenched by acrylamide, indicating that 8-ANS molecules preferentially bind at the protein surface. The fluorescence lifetime of the long-time component is independent of the protein and acrylamide concentration and may be related to protein-embedded dye molecules. Changes of the long lifetime component upon GdmCl-induced denaturation and unfolding of BCAB and alpha-HLA correlate well with overall changes of the protein conformation. The transition from native protein to the molten globule state is accompanied by an increase of the number of protein-embedded 8-ANS molecules, while the number of dye molecules located at the protein surface decreases. For the transition from the molten globule to the unfolded state was the opposite behaviour observed.

Acrylamide↗
PubMed1996

Influence of minor groove binders on the eukaryotic topoisomerase II cleavage reaction with 41 base pair model oligonucleotides.

This report deals with the cleavage reaction of calf thymus (CT) topoisomerase II with oligonucleotides containing one main cleavage site and adjacent binding sites for minor groove binders. The sequences of the oligonucleotides were derived from a pBR 322 sequence, which contains one main topoisomerase II cleavage site. The cleavage reaction was performed under increasing concentrations of minor groove binders and it showed characteristic inhibition dependences of topoisomerase II to the binding sites and to the binding length of the minor groove binders. The extension of the minor groove binder length on DNA from 4 to 10 base pairs (bp) by netropsin and bis-netropsin, respectively, causes a strong increase of the topoisomerase II cleavage inhibition. The same is observed by the introduction of a second minor groove binder sequence symmetrically positioned around the topoisomerase II main cleavage site. The combination of two different minor groove binders can lead to an increased topoisomerase II inhibition but also to a prevention of total inhibition as shown with chromomycin A3 and distamycin A at concentrations of 0.1 and 0.25 microM, respectively.

Animals↗
PubMed1996

Inhibition of restriction endonucleases by DNA sequence-reading ligands.

DNA sequence-reading bisquaternary ammonium heterocycles SN 6570, SN 6999, SN 6053, SN 6132, SN 6131, SN 18071 and the non-specific binders SN 6113, SN 5754, SN 6324, and SN 4094 influence the enzymatic activity of restriction endonucleases in different manners. A prerequisite for sequence-specific ligand interaction is a dAdT run of at least four base pairs. The sequence-specific binders inhibit the cleavage activity of restriction endonucleases EcoRI, SspI, and Dral with four and six dAdT base pairs in their restriction sites, while the activity of SalI and BamHI with less than four dAdT base pairs in their recognition motifs remains unaffected. On the contrary, the non-specific binding DNA ligands are incapable of suppressing the digestion for restriction nucleases under research. These results are in line with our footprint data. The inhibitory effect is independent of the number of cleavage sites in DNA and of whether the macromolecule exists in the ccc or lds conformation. Sequence specific binding of the ligand SN 6053 in close vicinity to the cleavage sites of restriction endonuclease Dral also interferes with enzyme inhibition.

Base Sequence↗
PubMed1996

Sequence-specific binding of antitumour bisquaternary ammonium heterocycles to DNA and inhibition of polymerase activity in vitro.

Ten bisquaternary ammonium heterocycles (BQA) active against experimental tumours were investigated for possible sequence-selective binding to DNA. Footprinting analyses indicated that several bound preferentially to dAdT runs consisting of at least four base pairs. Shortening of one or two spacer groups between the aromatic rings of the ligands (by replacement of CONH with NH) emerged as a prerequisite for sequence-specific binding. Other relevant factors concerned the overall shape of the ligands and the relative position of their positive charges. Footprinting plots evaluated for the BQA compound SN 6132 on the 167mer EcoRI-RsaI restriction fragment from plasmid pBR322 yielded the highest individual binding constant for the symmetrical base sequence AATTTAA, with approximate K(A) = 2.0 x 10(6)/M. Polymerase-catalysed syntheses of DNA and RNA in vitro were inhibited by all BQA derivatives, but the inhibition was much more pronounced with the sequence-specific binders SN 6999 and SN 6132 than with the non-specific ligand SN 6113.

Antineoplastic Agents↗
PubMed1996

Specific targeting of human papillomavirus type 16 E7 oncogene with triple-helix forming purine oligodeoxyribonucleotides.

Molecular mechanical calculations (computer modelling), optical DNA melting experiments and co-migration assay were used to assess stable helix formation at homopurine-homopyrimidine-rich target sites present in the human papillomavirus type 16 E7 oncogene (positions 656-673 on the genome map). The target sequence, either present in the E7 oncogene obtained by PCR technique or prepared from oligodeoxyribonucleotides (ODNs), can be specifically recognised by different 17-merpurine ODNs designed to form antiparallel or parallel triple helices. These "in vitro" experiments realised with rather long purine ODNs having a high degree of specificity, open the way for "in vivo" tests focused on E7 oncogene targeting and suppression.

Base Sequence↗
PubMed1995

Sequence specificity of DNA-psoralen photoproduct formation in supercoiled plasmid DNA (pUC19).

Supercoiled pUC19 DNA, photoreacted with psoralen derivatives (xanthotoxin (8-methoxypsoralen, 8-MOP), 4,5',8-trimethylpsoralen (TMP) and angelicin), influences the enzymatic activity of restriction enzymes in a different manner, although all the enzymes employed contain, within their recognition sites, suitable nucleic acid bases for photoproduct formation. The activity of the enzymes is strongly influenced by the photomodification of thymine residues within their recognition sites. 5'-TpA sequences favour intercalation as an essential prerequisite of the photoreaction, while 5'ApT sequences do not. This, in turn, influences photoproduct formation and the inhibition of the action of the restriction enzymes KpnI, SspI, DraI and RsaI, but not EcoRI and BamHI. The inhibitory effect is independent of the number of cleavage sites and also of whether monoaddition products or crosslinks are formed. Psoralen intercalation alone does not affect the activity of the restriction enzymes used.

Base Sequence↗
PubMed1995

Transcription specific differences visualized by fluorescence in situ hybridization pattern on interphase nuclei of different cell types.

Application of a "formamide free" and thus "material preserving" in situ hybridization technique using the cDNA of the myf3 gene revealed the following results: Human rhabdomyosarcoma cells, characterized by a high expression of myf3 show intensive hybridization signals in their interphase. RNase treatment prior to hybridization considerably reduces the size of this signals. In comparison, isolated nuclei of human lymphocytes in which no need for the expression of this gene exists, show barely hybridization signals. Correspondingly, RNase treatment had no effect on hybridization pattern at all. In conclusion an increased transcription efficiency of a cell type specific gene is accompanied by a higher hybridization accessibility in the corresponding cell nuclei.

Cell Nucleus↗
PubMed1994

Some new properties of DNA-YOYO-3 homodimer complexes revealed by electrophoresis and fluorescence lifetime measurements.

The DNA bis-intercalator oxazole homodimer (YOYO-3: 1,1'-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methy l- 2,3-dihydro-(benzo-1,3-oxazole)-2-methylidene]-quinolinium tetraiodide) specifically alters the electrophoretic pattern of covalently closed circular (CCC) DNA molecules. Thus, YOYO-3 seems to remove the CCC DNA supercoils and induces the appearance of additional bands by changing the linking number. It also promotes an unusual "star" activity for the restriction enzyme Hind III. Fluorescence lifetime measurements indicate that YOYO-3 is capable of binding to DNA by bis- and mono-intercalation.

Benzoxazoles↗