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Biomedical subjects

G Kaufmann

Publications and source records attributed to G Kaufmann.

At least 73 records · Page 4Linked to original sources
PubMed1984

Mammalian DNA enriched for replication origins is enriched for snap-back sequences.

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA, extruded DNA enriched for replication origins was obtained and denatured. Snap-back DNA, single-stranded DNA with inverted repeats (palindromic sequences), reassociates rapidly into stem-loop structures with zero-order kinetics when conditions are changed from denaturing to renaturing, and can be assayed by chromatography on hydroxyapatite. Origin-enriched nascent DNA strands from mouse, rat and monkey cells growing either synchronously or asynchronously were purified and assayed for the presence of snap-back sequences. The results show that origin-enriched DNA is also enriched for snap-back sequences, implying that some origins for mammalian DNA replication contain or lie near palindromic sequences.

Animals↗
PubMed1983

Aphidicolin arrest irreversibly impairs replicating simian virus 40 chromosomes.

The replicative DNA polymerase alpha is an intracellular target of aphidicolin. In vitro this drug inhibits DNA polymerase alpha reversibly. Yet, its in vivo effect on SV40 DNA replication, which depends on DNA polymerase alpha, was found to be irreversible. Thus, exposure of infected cells to aphidicolin led to a progressive loss in their ability to incorporate [3H]dT into viral DNA in a subsequent pulse without drug. This loss was time-dependent (t1/2 at 37 degrees C at 2 microgram/ml of drug was approximately 20 min) and increased with drug concentration. Likewise, replicating SV40 DNA, pulse-labeled prior to exposure, lost the ability to mature into form I DNA upon removal of the drug. No degradation of replicating SV40 DNA molecules was detected by neutral sucrose gradient analysis during or up to 1 h after aphidicolin exposure. However, longer incubations resulted in breakdown of the arrested replicative intermediate, concomitant with the resumption of viral DNA synthesis. Origin-synchronized SV40 replicons were less affected by exposure to aphidicolin than were ongoing replicons, as judged from comparing recoveries of tsA replicons from 40 degrees C restriction, with or without the drug. The data indicate that replicating SV40 chromosomes become selectively impaired during aphidicolin arrest and prevent thereby the initiation of new replication rounds, perhaps by occupying fixed nuclear replication sites.

Animals↗
PubMed1983

Influence of a 9-double bond on stereospecific microbial 4,5-reductions.

By stereospecific microbial reduction with Rhodosporidium rubrum or Rhodotorula glutinis, 17 alpha-cyano-methyl-4-estren-17 beta-ol-3-one was metabolized to 17 alpha-cyanomethyl-5 alpha-estrane-3 beta,17 beta-diol (50%) and 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha,17 beta-diol (30%). By Clostridium paraputrificum the same substrate was reduced stereospecifically to 17 alpha-cyanomethyl-5 beta-estrane-3 alpha, 17 beta-diol (70%). When the corresponding 9-dehydrogenated compound 17 alpha-cyanomethyl-4,9-estradien-17 beta-ol-3-one (STS 557, a new progestagen) was fermented, yeasts failed in 5 alpha-reducing the 4-double bond. Still Clostridium paraputrificum formed the expected 5 beta-reduced metabolite 17 alpha-cyanomethyl-5 beta-estr-9-ene-3 alpha,17 beta-diol (60%). Structures were elucidated by n.m.r. and mass spectra and partly by circular dichroism. By oxidation of the metabolites, the corresponding 3-oxo compounds 17 alpha-cyanomethyl-5 alpha-estran-17 beta-ol-3-one, 17 alpha-cyanomethyl-5 beta-estran-17 beta-ol-3-one and 17 alpha-cyanomethyl-5 beta-estr-9-en-17 beta-ol-3-one were prepared. The evident influence of the 9-double bond on reduction of the 4-en-3-oxo compound STS 557 preventing 5 alpha-reduction but permitting 5 beta-reduction is discussed in view of the distinctly diminished metabolism of this progestagen in mammals.

Clostridium↗
PubMed1983

Studies on biotransformation of STS 557.

Transformation of STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one) by female rat liver microsomes demonstrates a lower transformation rate in comparison with the analogous compound without 9-double bond: 17 alpha-cyanomethyl-19-nortestosterone, and the basic substance: 19-nortestosterone. 17 alpha-Cyanomethyl-estra-1, 3, 5(10), 9(11)-tetraene-3, 17 beta-diol, 17 alpha-cyanomethyl-11 beta, 17 beta-dihydroxy-estra-4, 9-dien-3-one, and tentatively 17 alpha-cyanomethyl-6 alpha, 17 beta-dihydroxy-estra-4, 9-dien-3-one were identified as metabolites. Microbial model investigations with species known to hydrogenate the 4-double bond in 4-en-3-oxo steroids stereospecifically to 5 alpha H- or 5 beta H-metabolites indicate 5 alpha-hydrogenation to be prevented in STS 557 by the 9-double bond, whereas 5 beta-hydrogenation is not affected. Isolation and characterization of metabolites from beagle dog and rat urine following administration of 3H-STS 557 revealed the following pathways of biotransformation: Hydroxylation in different positions of the steroid molecule, aromatization of ring A, hydrogenation of a double bond, simultaneous hydroxylation and hydrogenation, and alteration of the 17 alpha-side chain with loss of nitrogen.

Animals↗
PubMed1983

Receptor binding of STS 557.

Binding of the new progestagen STS 557 (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4, 9-dien-3-one) to progesterone receptors was investigated by competition experiments using cytosol preparations from chick oviduct, rat uterus, rabbit uterus and human endometrium. A high competitive potency of STS 557 was found in the mammalian systems used. The binding affinity was about 20% that of progesterone. Binding to cytosol receptors of the chick oviduct was two orders lower. Five derivatives of STS 557 were compared with respect to progesterone receptor binding by using rabbit uterus cytosol. The hydrogenated 3-oxo compounds exhibited receptor affinities similar to the parent compound as for as the planarity of the steroid ring system was sustained. 5 beta-H compounds were nearly inactive. Additionally, binding to the androgen receptor of rat prostate cytosol was measured. STS 557 and its planar derivatives were found to be effective competitors for 5 alpha-dihydrotestosterone (DHT) exhibiting binding affinities about one fifth that of DHT. The findings are discussed with respect to the chemical structures and the in vivo effects of STS 557.

Animals↗
PubMed1982

An oligoribonucleotide polymerase from SV40-infected cells with properties of a primase.

A transient decaribonucleotide (iRNA) is covalently linked to nascent eukaryotic DNA chains at their 5' end. Searching for the putative iRNA polymerase (primase), we detected in extracts from SV40-infected cells a DNA-dependent incorporation of UMP residues from UTP into free and DNA linked deca- or similarly sized ribonucleotides. Denatured salmon sperm DNA served as the standard template in this reaction. SV40 FIII DNA was also an effective template, SV40 FII DNA was ineffective while FI yielded mainly free decaribonucleotides. The incorporation depended on the other rNTPs and was resistant to high concentrations of alpha-amanitin and rifamycin AF/013, drugs inhibitory to RNA polymerases I, II and III. The results implicate the decaribonucleotide polymerase in the priming of nascent DNA chains and suggest that the unique size of iRNA is encoded within its primase.

Amanitins↗
PubMed1982

Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest.

To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40 degrees, to complete ongoing replication and returned to 32 degrees, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40 degrees without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (alpha-32p)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI- restricted SV40 DNA, demonstrating partial replicon synchronization. The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.

Animals↗
PubMed1982

A laboratory method for testing the volatility from aqueous solution: first results and comparison with theory.

The mobility of a chemical in the environment is often determined by its volatilization from aqueous solution, especially if it shows poor water solubility and/or high vapor pressure. A small laboratory apparatus is described which allows the simulation of the volatilization process from a stirred water body with well-defined wind velocities and water temperatures. In experiments with naphthalene, pentachlorophenol, trichloroethene, and di(2-ethylhexyl)phthalate, substance concentrations in the water body decrease exponentially, in accordance with theory. Half-residence times are found to be independent of wind velocity (v less than 1 m sec-1) or air humidity, but are strongly dependent on temperature. The measured substance fluxes through the water surface are comparable with theoretical values. In the case of pentachlorophenol, which partly dissociates in aqueous solution, the volatility is controlled by the pH value of the solution.

Diethylhexyl Phthalate↗
PubMed1982

Bacteriophage T4-induced anticodon-loop nuclease detected in a host strain restrictive to RNA ligase mutants.

The fate of host tRNAs during T4 bacteriophage infection was investigated with Escherichia coli CTr5x, the only known host strain that is restrictive to RNA ligase and polynucleotide kinase mutants. Three CTr5x tRNA species were cleaved during infection. One was leucine tRNA1, which was cleaved in the extra arm, as reported elsewhere for E. coli B infected with bacteriophage T2 or T4. The other two were specific to E. coli CTr5x and were not cleaved in various other hosts. One of the cleaved CTr5x-specific tRNAs had an anticodon sequence of the E. coli B "major" isoleucine tRNA but otherwise little sequence homology. Both CTr5x-specific tRNAs were cleaved by a distinct T4-induced endonuclease, other than that of leucine tRNA1, because the CTr5x-specific cleavages (i) were induced later in infection, (ii) persisted with a T4 mutant deficient in leucine tRNA1 endonuclease, and (iii) occurred in the anticodon loop. The specific manifestation of the anticodon-directed endonuclease activity in T4-infected E. coli CTr5x suggests roles for RNA ligase and polynucleotide kinase in processing of host tRNA species.

Anticodon↗
PubMed1981

[Ambulatory treatment of heart rhythm disorders].

Partial suppression of symptomatic arrhythmias is feasible in most instances. Suitability of drugs for long-term treatment depends on their side effects, costs and action duration. Discrepancies between elimination half life and duration of action are described. Perhaps the most important shortcoming in long-term suppression of potentially dangerous arrhythmias is the short-lived action of most available drugs. In a once-daily regimen with allowance for occasional omission, a drug action duration of more than 48 hours is desirable. This applies only to perhexiline, nadolol, amiodarone, digitoxin, and digoxin in elderly patients.

Ambulatory Care↗