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Biomedical subjects

G Burckhardt

Publications and source records attributed to G Burckhardt.

At least 19 recordsLinked to original sources
PubMed1998

Binding of 2-azaanthraquinone derivatives to DNA and their interference with the activity of DNA topoisomerases in vitro.

We have investigated the binding ability to DNA of compounds belonging to the 2-azaanthraquinone-type structure and have examined the effect on the activity of DNA gyrase as well as on mammalian topoisomerases in vitro. Using different biophysical techniques it was found that one of these ligands, 9-((2-dimethylamino)ethyl)amino)-6-hydroxy-7-methoxy-5, 10-dihydroxybenzo[g]isoquinoline-5,10-dione (TPL-I), is an intercalating DNA binding agent, whereas the parent compound tolypocladin (TPL) and a derivative (TPL-II) showed almost no similar affinity to DNA. CD measurements demonstrated a significant and selective binding tendency of TPL-I to alternating purine/pyrimidine sequences with some preference for poly(dA-dT). poly(dA-dT). Tm values were increased of the ligand complex with the alternating AT-containing duplex polymer. The binding to various DNAs was characterized by CD and visible absorption spectral changes. From the latter, different binding constants of 6.2 x 10(5) and 1.5 x 10(5) M-1 were obtained for poly(dA-dT).poly(dA-dT) and poly(dA). poly(dT), respectively. Sedimentation measurements with supercoiled pBR322 plasmid DNA clearly indicated an intercalative binding mechanism associated with an unwinding angle of about 18 degrees. These results suggest that the intercalative binding of TPL-I is promoted by the 2-(dimethylamino)ethylamino group substituted on carbon 9 of the anthraquinone system. The cytotoxic compound TPL-I, but not TPL or TPL-II, effectively inhibited the DNA supercoiling reaction of DNA gyrase and the activity of mammalian topoisomerases I and II as measured by the relaxation assay. TPL-I affects the cleavage reaction of topoisomerases on a single site located in alternating purine-pyrimidine sequence regions. The inhibitory potency of TPL-I can be ascribed to a blocking of cleavage sites on the DNA substrate, which correlates with the sequence preference of the ligand.

Anthraquinones↗
PubMed1998

Characterization of the chloride conductance in porcine renal brush-border membrane vesicles.

The chloride conductance in brush-border membrane vesicles prepared from pig kidney cortex was investigated using a light-scattering assay, anion-diffusion-potential-dependent Na+-D-glucose cotransport and 36Cl- influx. K+-diffusion-potential-driven salt exit from, or entry into, the vesicles was slow in the presence of gluconate, SO42- and F-, intermediate with Cl- and Br-, and fast with I-, NO3-, and SCN-. Stimulation of Na+-D-glucose uptake followed a similar anion sequence. Conductive Cl- flux had a low activation energy and was inhibited by suphhydryl reagents, the stilbene disulphonates 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonate (SITS) and 4, 4'-diisothiocyanato-2,2'-disulphonate (DIDS), and the arylaminobenzoates diphenylamine-2-carboxylic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Intravesicular Ca2+ and extravesicular nucleotides were without effect on conductive Cl- flux. These characteristics tentatively exclude some known Cl- channels and leave members of the ClC family as possible candidates responsible for the Cl- conductance in brush-border membranes.

Animals↗
PubMed1998

Endosomes: another extra-mitochondrial location of type-1 porin/voltage-dependent anion-selective channels.

Endocytotic vesicles (EV) isolated from rat renal cortex were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting. A monoclonal antibody against human type-1 porin (31 kDa) detected a strong band of 31 kDa. The same antibody has been used as the primary antibody in indirect immunocytochemistry. Light microscopy of cryostat sections of rat renal cortex showed a heavy staining of EV underneath the brush-border membrane. Electron microscopy was performed by "preembedding immunogold staining" of rat renal cortex, the sections of which showed an extensive labelling of EV with gold particles. These results demonstrate that the expression of type-1 porin is not restricted to outer mitochondrial membranes. The biological function of endosomal type-1 porin has as yet to be ascertained.

Animals↗
PubMed1998

S3226, a novel inhibitor of Na+/H+ exchanger subtype 3 in various cell types.

Inhibition of Na+/H+ exchange (NHE) subtypes has been investigated in a study of the mouse fibroblast L cell line (LAP1) transfected with human (h) NHE1, rabbit (rb) NHE2, rat (rt) or human (h) NHE3 as well as an opossum kidney cell line (OK) and porcine renal brush-border membrane vesicles (BBMV). S3226 ¿3-[2-(3-guanidino-2-methyl-3-oxo-propenyl)-5-methyl-phenyl]-N-isopro pylidene-2-methyl-acrylamide dihydro-chloride¿ was the most potent and specific NHE3 inhibitor with an IC50 value of 0.02 micromol/l for the human isoform, whereas its IC50 value for hNHE1 and rbNHE2 was 3.6 and approximately = 80 micromol/l, respectively. In contrast, amiloride is a weak NHE3 inhibitor (IC50>100 micromol/l) with a higher affinity to hNHE1 and rbNHE2. Cariporide (4-isopropyl-3-methylsulphonyl-benzoyl-guanidine methane-sulphonate), which has an IC50 for NHE3 of approximately 1 mmol/l, is a highly selective NHE1 inhibitor (0.08 micromol/l). Therefore, S3226 is a novel tool with which to investigate the physiological and pathophysiological roles of NHE3 in animal models.

Amiloride↗
PubMed1997

Expression cloning and characterization of a renal organic anion transporter from winter flounder.

The cDNA coding for a renal p-aminohippurate (PAH) transporter from winter flounder (Pseudopleuronectes americanus), designated fROAT, was cloned by functional expression in Xenopus laevis oocytes. fROAT is approximately 2.8 kbp in length and encodes a protein of 562 amino acids, related to the rat renal organic anion transporter ROAT1/OAT1 and the organic cation transporters OCT1 and OCT2. In oocytes, fROAT mediated probenecid-sensitive PAH uptake, with a Km for PAH of about 20 microM, and inhibited by external glutarate (GA) (1 mM). The functional characteristics suggest that fROAT is the basolateral PAH/dicarboxylate exchanger of the flounder kidney.

Amino Acid Sequence↗
PubMed1997

NH4+ conductance in Xenopus laevis oocytes. I. Basic observations.

Current-clamp and voltage-clamp techniques were used to study the effects of NH4+ on the cell membrane conductance in Xenopus laevis oocytes. Superfusing the oocytes with NH4Cl resulted in a depolarization of the oocyte's cell membrane potential and, at a clamp potential of -70 mV, in an inward current. The magnitude of the inward current was proportional to the NH4Cl concentration in the extracellular solution and on membrane potential. The reversal potential, Erev , was -35.5 +/- 11.6 mV under control conditions and -3.1 +/- 11.0 mV (n = 19) in the presence of NH4Cl (10 mmol/l). Superfusion of the oocytes with nominally Ca2+-free solution affected the NH4Cl-evoked response only marginally. Replacement of extracellular Na+ by N-methyl-D-glucamine+ markedly reduced, but did not eliminate, the NH4Cl-sensitive current and shifted the reversal potential to more negative potentials. The NH4Cl-induced current was substantially inhibited by 0.1 mmol/l flufenamate, and was less affected by blockers of the endogenous K+ conductance, Ba2+ and isosorbiddinitrate (ISDN). The results are compatible with the activation of a conductance by NH4Cl for Na+ and NH4+. The mechanism by which NH4Cl activates the conductance remains unknown.

Animals↗
PubMed1997

Absence of vacuolar H+-ATPases from rat small intestine brush-border membranes.

Rat small intestinal brush-border membrane (BBM) vesicles were solubilized to test for the presence of an intravesicular H+-ATPase. Several detergents that in rat renal BBM vesicles revealed a bafilomycin-sensitive H+-ATPase failed to expose a similar ATPase in small intestinal vesicles. Antibodies against the 31-kDa and 70-kDa H+-ATPase subunits labelled respective antigens in the BBM of renal proximal tubules, but not in the BBM of enterocytes. The results demonstrate that a bafilomycin-sensitive, vacuolar-type H+-ATPase is absent from the BBM of enterocytes and, hence, cannot contribute to H+ secretion.

Animals↗
PubMed1997

Hairpin polyamides that use parallel and antiparallel side-by-side peptide motifs in binding to DNA.

Pt-bis-netropsin is a synthetic sequence-specific DNA-binding ligand comprizing two netropsin-like fragments which are linked in a tail-to-tail manner via a cis-diammineplatinum (II) residue. The CD studies and thermodynamic characterization of the DNA-binding properties exhibited by this compound reveal that it forms two types of complexes with poly[d(AT)].poly[d(AT)] and DNA oligomers containing nucleotide sequences 5'-CC(TA)n CC-3', with n = 4, 5 and 6. The first type corresponds to the binding of Pt-bis-netropsin in the extended conformation and is characterized by the saturating ratio of one bound Pt-bis-netropsin molecule per 9 AT-base pairs. The second type of the complex corresponds to the binding of Pt-bis-netropsin to DNA in the folded hairpin form. The binding approaches saturation level when one Pt-bis-netropsin molecule is bound per four or five AT-base pairs. The hairpin form of Pt-bis-netropsin complex is built on the basis of parallel side-by-side peptide motif which is inserted in the minor DNA groove. The CD spectral profiles reflecting the binding of Pt-bis-netropsin in the hairpin form are different from those observed for binding of another bis-netropsin with the sequence Lys-Gly-Py-Py-Gly-Gly-Gly-Py-Py-Dp, where Py is a N-propylpyrrole amino acid residue and Dp is a dimethylaminopropylamino residue. The hairpin form of this bis-netropsin is formed on the basis of antiparallel side-by-side peptide motif. The CD spectra obtained for complexes of this polyamide in the hairpin form with poly[d(AT)].poly[d(AT)] exhibit positive CD band with a peak at 325 nm, whereas the CD spectral profiles for the second complex of Pt-bis-Nt with poly[d(AT)].poly[d(AT)] and short DNA oligomers have two intense positive CD bands near 290 nm and 328 nm. This reflects the fact that two bis-netropsins use different structural motifs on binding to DNA in the hairpin form.

Amino Acid Sequence↗
PubMed1997

DNA binding studies and influence on the activity of DNA topoisomerases of bis-netropsins: different effects of analogs containing cis and trans ethylene linkers.

Binding to DNA and synthetic duplex polymers of two bifunctional netropsins and effects on supercoiled plasmid DNA as well as their inhibitory potency on DNA topoisomerases have been investigated. Characteristic differences were found in the DNA binding properties of the two bis-netropsins containing a cis and trans tether as reflected by CD, thermal melting and sedimentation measurements. CD results indicate, that the bis-netropsins interact with DNA by a two-step binding mode depending on the ligand concentration. The trans bis-netropsin may form stable complexes with different DNA's at high salt concentration, whereas for cis bis-netropsin DNA complexes the second binding step is completely abolished. The variations in the DNA binding ability of trans and cis bis-netropsin show a close relationship to the differences observed in their inhibitory effects on DNA topoisomerases. It appeared that trans bis-netropsin more strongly blocks topoisomerase activity than the cis isomer and represents the most potent inhibitor of DNA gyrase. Differences in the DNA. binding ability of the bis-netropsins and their inhibitory potency on topoisomerase activity are explained in terms of bidentate and monodentate binding mode of the trans and cis isomer, respectively.

Binding, Competitive↗
PubMed1996

The detection of B-form/A-form junction in a deoxyribonucleotide duplex.

The transition of the 14-meric deoxyoligonucleotide duplex d-(ACCCCCTTTTTTTG).d-(CAAAAAAAGGGGGT) from the B- to the A-conformation in water/trifluorethanol (TFE) solution was studied with the use of circular dichroism. An increase in the fraction of TFE induces a two-step B-A transition. In the first step, up to 73% TFE, the A-form is generated from the GC-rich part; in the second step, 73-82% TFE, the AT-rich part shifts to the A-form. By this we suggest the existence of a B/A junction near 73% TFE. Emergence of the B/A junction has been directly confirmed with the use of distamycin A and netropsin, ligands known to selectively bind to AT stretches of B-DNA. It can be shown that both ligands suppress formation of the A-form in the B-philic part. The free energy value for the B/A junction was estimated to be 2.1 kcal/mol, which agrees well with known data for polymeric DNAs. The obtained results may have biological relevance in connection with recently published x-ray data about the occurrence of the B/A junction in the complex of DNA with reverse transcriptase of HIV.

Base Composition↗
PubMed1996

Reversal of the Z- to B-conformation of poly(dA-dT) center dot poly(dA-dT) induced by netropsin and distamycin A.

Poly(dA-dT) center dot poly(dA-dT) which adopts the Z-form at 5 M NaCl in presence of 95 mM Ni2+ions is reversed to the B-conformation by the nonintercalating drugs netropsin (Nt) and distamycin A (Dst). The drug-induced reversal from the Z-to B-form of poly(dA-dT) center dot poly(dA-dT) is evidenced by CD spectral changes at characteristic wavelengths around 295 nm and 248 nm. The drug-induced conformational transition is accompanied by a slow kinetic process. The results suggest the preference of these AT-specific drugs for the B-form and the inability of Nt and Dst to form a stable complex with the Z-form of poly(dA-dT) center dot poly(dA-dT).

Bacterial Proteins↗
PubMed1995

Ecto-adenosinetriphosphatase in rat small intestinal brush-border membranes.

Antibodies against the holo ecto-adenosinetriphosphatase (ATPase) of rat liver and antibodies against COOH-terminal peptides of the long isoform of this enzyme reacted in Western blots with a 105-kDa band from small intestinal brush-border membranes. Indirect immunofluorescence revealed reactive proteins predominantly at the apical surface of enterocytes with some staining of basolateral membranes and of vascular endothelium. Similar results were obtained with monoclonal antibodies against HA4, a protein from rat liver closely related to the ecto-ATPase. Since these results suggested the presence of an ecto-ATPase, ATP hydrolysis was studied in intact, right-side-out brush-border membrane vesicles. Nearly half of ATP hydrolysis was caused by alkaline phosphatase (AP). Besides purine and pyrimidine trinucleotides, AP also hydrolyzed ADP, AMP, pyrophosphate, and 4-nitrophenylphosphate. Inactivation of AP by cleavage of its membrane anchor and by removal of the Zn2+ necessary for its function left the ecto-ATPase that was activated by Ca2+ and Mg2+ and hydrolyzed purine and pyrimidine trinucleotides and dinucleotides, but not AMP, pyrophosphate, and 4-nitrophenylphosphate. These features are characteristic of an ATP diphosphohydrolase (EC 3.6.1.5, also called apyrase). The physiological role of the small intestinal ecto-ATPase may be the degradation of nutrient nucleotides.

Adenine Nucleotides↗
PubMed1994

Expression of rat renal Na+HCO3-cotransporter in Xenopus laevis oocytes.

The goal of this study was to determine whether Na+HCO3- cotransport from rat renal cortex can be functionally expressed in Xenopus laevis oocytes. Using a two electrode voltage-clamp device, HCO(3-)-dependent currents were determined as the difference in current measured in a nominally HCO(3-)-free buffer, pH 7.5 and a buffer with 30 mmol/l HCO3-, pCO2 5.33 kPa, pH 7.5. The size of renal cortex mRNA required to express maximum HCO(3-)-dependent current was 2-3 kb. The expressed current depended on Na+ and was sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and acetazolamide (each 1 mmol/l), inhibitors known to block the native Na(+)HCO3- cotransporter in several tissues including rat proximal tubule. Water-injected oocytes did not show any measurable response either to increased HCO3- nor to Na(+)-free perfusion or to the inhibitors, indicating the absence of an endogenous electrogenic HCO3-transporter. Our data indicate that the rat renal mRNA of 2-3 kb size contains the message for the Na+HCO3-cotransporter.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Modulation of DNA supercoiling by interaction with netropsin and other minor groove binders.

The assay of DNA unwinding by ethidium, followed by sedimentation velocity techniques, was applied to complexes of supercoiled plasmid DNA with different non-intercalating drugs which strongly and sequence-specifically bind to DNA. Compared with the behaviour of naked DNA, most of the complexes exhibit an increase in the critical EB/nucleotide binding ratio associated with the principal minimum in the sedimentation profile. Using netropsin (Nt) as the paradigm of the minor groove binders investigated, the drug-induced alterations in various structural parameters of both the relaxed and supercoiled form of DNA are described. Whereas winding number, helical repeat (both being defined with reference to a surface normal), and linking number of the superhelical DNA remain constant in our experiments, its twist number, surface twist, number of superhelical turns as well as the absolute values of linking number difference, superhelix density, and writhing number increase on binding of Nt. Correspondingly, compared with the naked relaxed DNA a higher linking number (or twist number, or winding number), a higher average duplex winding angle and a lower helical repeat have to be assigned to the relaxed Nt-DNA complex. The various minor groove binders investigated were found to differ considerably in their efficiency to alter the structure of supercoiled DNA.

Base Sequence↗
PubMed1993

Studies on the ability of minor groove binders to induce supercoiling in DNA.

The effect of various non-intercalating minor groove binders on closed circular DNA in the presence of topoisomerase I has been studied by means of agarose gel electrophoresis. Analogues of the netropsin series (lexitropsins) and SN-6999 effectively produce positive supercoils, as indicated by analysis of the topoisomers in the presence of chloroquine and the evaluated linking number changes. Analogues of the distamycin series are less effective, and bisquaternary heterocycles, as well as DAPI and pentamidine, were found to be ineffective ligands. The large differences observed in the ability of minor groove binders to induce positive supercoils are discussed.

DNA Topoisomerases, Type I↗
PubMed1993

Binding to DNA of selected lexitropsins and effects on prokaryotic topoisomerase activity.

The binding behaviour toward DNA of some minor groove binders related to distamycin was studied by means of circular dichroism. In addition their influence on the activity of topoisomerases isolated from Streptomyces noursei has been investigated. The monocationic imidazole containing ligands (lexitropsins) show a decreased affinity to AT pairs but an increased affinity to GC pairs which contrasts the AT-preferred binding of Dst-2 and Dst-3. For the monocationic triimidazole containing lexitropsin the affinity for GC over AT pairs was most pronounced. It was also found that the imidazole containing lexitropsins are inhibitors of topoisomerases. These minor groove binders interfere more strongly with the DNA gyrase activity than with the prokaryotic topoisomerase I. Our results indicate that Dst-3 most effectively inhibits gyrase and topoisomerase I activity. However, the inhibitory effect is neither related to the base pair specificity nor to the binding strength of different ligands. The mechanism of interference of minor groove binders with topoisomerase activity is more complex. It is considered that different factors, such as the nature of the ligand together with their DNA binding parameters and the target sequences of the enzymes play a role in the inhibitory effects of minor groove binders.

Animals↗