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Biomedical subjects

F Eckstein

Publications and source records attributed to F Eckstein.

At least 253 records · Page 14Linked to original sources
PubMed1990

The metabolism of 3'-azido-2',3'-dideoxyguanosine in CEM cells.

When CEM cells were incubated with [3H]-labeled 3'-azido-2',3'-dideoxyguanosine (AzddGuo), the isotope was largely recovered as ribo- and deoxyribonucleotides in the acid soluble fraction of CEM cells, due to extensive catabolism of AzddGuo and recycling of the guanine formed by purine nucleoside phosphorylase. Only 10% was found as the AzddGuo nucleotides, with the diphosphate of AzddGuo as the dominating nucleotide at all time points. Thus nucleoside diphosphate kinase was rate limiting for the formation of AzddGuo triphosphate, responsible for the toxic and antiviral activity of the nucleoside. Inhibition of de novo deoxyribonucleotide synthesis with hydroxyurea increased the phosphorylation of AzddGuo twofold.

Antiviral Agents↗
PubMed1990

High-efficiency oligonucleotide-directed plasmid mutagenesis.

A number of single- and double-base substitutions have been introduced into either the polylinker region or the lacZ gene in the plasmid vector pUC19. The efficiencies of these changes upon transfection of TG-1 bacterial cells were generally 70-80%. A strategy has been devised by which the wild-type DNA can be selectively destroyed. It is primarily based on the resistance of phosphorothioate internucleotide linkages to some restriction enzymes. A mismatch oligonucleotide is introduced into a gapped region and the gap is filled using three deoxynucleoside 5'-triphosphates and one deoxynucleoside 5'-[alpha-thio]triphosphate. Reaction with a restriction enzyme that is unable to hydrolyze phosphorothioates ensures that the DNA containing the mismatch oligonucleotide is only nicked. Concomitantly, the DNA that does not contain the desired mutation is linearized. Subsequent reactions with an exonuclease and DNA polymerase I yield mutant homoduplex DNA for transfection.

Base Sequence↗
PubMed1990

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Amino Acid Sequence↗
PubMed1989

Hydroxyurea increases the phosphorylation of 3'-fluorothymidine and 3'-azidothymidine in CEM cells.

The triphosphates of the nucleoside analogues 3'-azidothymidine and 3'-fluorothymidine inhibit reverse transcriptase and are of therapeutic interest for the treatment of retrovirus infections. At equimolar concentrations 3'-fluorothymidine was more effectively transformed to the triphosphate by human CEM cells than azidothymidine which mainly accumulates as the monophosphate. Hydroxyurea, a drug that inhibits de novo synthesis of deoxyribonucleotides, considerably increased the ability of cells to phosphorylate both analogues. Addition of as little as 50 microM hydroxyurea decreased the amount of dideoxynucleoside required to attain a given intracellular concentration of its triphosphate by an order of magnitude. Hydroxyurea is known to shift the balance of substrate cycles between natural deoxynucleosides and their 5'-phosphates in the direction of synthesis and thereby to increase the import and intracellular phosphorylation of the nucleoside. The present results demonstrate a similar effect for the two analogues and raise the possibility of using this effect in therapy.

Antiviral Agents↗
PubMed1989

Incomplete primer extension during in vitro DNA amplification catalyzed by Taq polymerase; exploitation for DNA sequencing.

Polyacrylamide gel electrophoresis of DNA fragments obtained by the polymerase chain reaction using Taq polymerase revealed the presence of multiple fragments shorter than the expected product. These abortive extension products were observed even when analysis by agarose gel electrophoresis showed only a single band. The production of prematurely terminated fragments can be exploited for the sequencing of PCR products if phosphorothioate groups are incorporated base specifically during the reaction in the presence of two oligonucleotide primers, one of which is 5'-32P-labeled. The addition of snake venom phosphodiesterase to the reaction mixture after completion of the amplification cycles digests each fragment from the 3'-end to a phosphorothioate group so that the sequence can be read by polyacrylamide gel electrophoresis.

Base Sequence↗
PubMed1989

Purine and pyrimidine nucleotides potentiate activation of NADPH oxidase and degranulation by chemotactic peptides and induce aggregation of human neutrophils via G proteins.

Whereas the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), induced NADPH-oxidase-catalyzed superoxide (O2-) formation in human neutrophils, purine and pyrimidine nucleotides per se did not stimulate NADPH oxidase but enhanced O2- formation induced by submaximally and maximally stimulatory concentrations of fMet-Leu-Phe up to fivefold. On the other hand, FMet-Leu-Phe primed neutrophils to generate O2- upon exposure to nucleotides. At a concentration of 100 microM, purine nucleotides enhanced O2- formation in the effectiveness order adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]) greater than ITP greater than guanosine 5'-O-[3-thio]triphosphate (GTP[gamma S]) greater than ATP = adenosine 5'-O-[2-thio]triphosphate (Sp-diastereomer) = GTP = guanosine 5'-O-[2-thio]diphosphate (GDP[beta S] = ADP greater than adenosine 5'-[beta, gamma-imido]triphosphate = adenosine 5'-O-[2-thio]triphosphate] (Rp-diastereomer). Pyrimidine nucleotides stimulated fMet-Leu-Phe-induced O2- formation in the effectiveness order uridine 5'-O-[3-thio]triphosphate (UTP[gamma S]) = UTP greater than CTP. Uracil (UDP[beta S]) = uridine 5'-O[2-thio]triphosphate (Rp-diastereomer) (Rp)-UTP[beta S]) = UTP greater than CTP. Uracil nucleotides were similarly effective potentiators of O2- formation as the corresponding adenine nucleotides. GDP[beta S] and UDP[beta S] synergistically enhanced the stimulatory effects of ATP[gamma S], GTP[gamma S] and UTP[gamma S]. Purine and pyrimidine nucleotides did not induce degranulation in neutrophils but potentiated fMet-Leu-Phe-induced release of beta-glucuronidase with similar nucleotide specificities as for O2- formation. In contrast, nucleotides per se induced aggregation of neutrophils. Treatment with pertussis toxin prevented aggregation induced by both nucleotides and fMet-Leu-Phe. Our results suggest that purine and pyrimidine nucleotides act via nucleotide receptors, the nucleotide specificity of which is different from nucleotide receptors in other cell types. Neutrophil nucleotide receptors are coupled to guanine-nucleotide-binding proteins. As nucleotides are released from cells under physiological and pathological conditions, they may play roles as intercellular signal molecules in neutrophil activation.

Cell Aggregation↗
PubMed1989

Phosphorothioates in molecular biology.

The observation that phosphorothioate analogues of the nucleoside triphosphates are substrates for DNA- and RNA-polymerases has proven a boon for the molecular biologist. As these phosphorothioate-containing polymers are stable to degradation by nucleases and the sulfur atom confers many favourable chemical properties, several applications in molecular biology have been developed, including new methods for site-directed mutagenesis and DNA sequencing.

DNA↗
PubMed1988

Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides.

The Rp- and Sp-diastereomers of the phosphorothioate-containing oligonucleotide d[ApAp(S)ApA] have been synthesized. They and the tetramer d[ApApApA] were tested as substrates for staphylococcal nuclease, DNase II and spleen phosphodiesterase. For digestions with DNase I these oligonucleotides were converted to the 5'-phosphorylated derivates. The reactions with the nucleases were analysed by HPLC. The phosphorothioate groups of both diastereomers were resistant to the action of staphylococcal nuclease, DNase I and DNase II. While the phosphorothioate group of the Rp-diastereomer was resistant to the action of spleen phosphodiesterase, the Sp-diastereomer was hydrolysed at an estimated rate 1/100 the rate of cleavage of the unmodified tetramer. The presence of the phosphorothioate group in the center of the molecule affected the rate of hydrolysis of neighbouring phosphate groups for some enzymes. In particular, very slow release of 3'-dAMP from the Rp-diastereomer occurred on incubation with staphylococcal nuclease but the Sp-diastereomer was completely resistant. DNase II produced 3'-dAMP quite rapidly from both diastereomers of d[ApAp(S)ApA] and DNase I released 5'-dAMP from both diastereomers of d[pApAp(S)ApA] only slowly.

Animals↗
PubMed1988

Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates.

The direct sequencing of DNA generated by the polynucleotide chain reaction, via the incorporation of phosphorothioate nucleotides and followed by treatment with an alkylating reagent that cleaves specifically at the phosphorothioate positions, is described. The Taq polymerase used in the amplification reaction incorporates the Sp-diastereomer of the deoxynucleoside 5'-O-(1-thiotriphosphates) as efficiently as the natural nucleotides. Chemical degradation of the phosphorothioate-containing DNA fragment can be performed with either 2-iodoethanol or 2,3-epoxy-1-propanol. The higher reactivity of 2,3-epoxy-1-propanol allows less reagent to be used to obtain the same amount of degradation as with 2-iodoethanol.

Base Sequence↗
PubMed1988

RNA mediated formation of a phosphorothioate diester bond.

Previous results showed that multimeric, tandemly sequence-repeated forms of satellite tobacco ringspot virus RNA of the encapsidated polarity (STobRV (+)RNA) autolytically process at a specific phosphodiester bond, the junction. Substituting a phosphorothioate diester bond for the STobRV (+)RNA junction drastically slowed autolytic processing. Here we show that for the complementary STobRV (-)RNA, in contrast, replacing sets of phosphodiester bonds with phosphorothioate diester bonds, even at the junction, did not greatly slow autolytic processing or spontaneous ligation, the usual reactions of the unmodified RNA. In the ligation reaction STobRV (-)RNA directed the formation of an ApG phosphorothioate diester bond.

Base Sequence↗
PubMed1988

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments.

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1988

A role for divalent cations in specifying the start site for transcription from chromatin templates in vitro.

An assay that employs nucleoside 5'-O-(2-thiotriphosphates) was used to detect initiation of mouse mammary tumor virus (MMTV) RNA chains in preparations of isolated nuclei from cultured rat hepatoma cells containing stably integrated proviruses. RNA chains initiated with adenosine 5'-O-(2-thiotriphosphate), guanosine 5'-O-(2-thiotriphosphate), or uridine 5'-O-(2-thiotriphosphate) were separated from the remaining RNA by mercury-Sepharose column chromatography and analyzed for correctly initiated RNA chains with a T1 nuclease protection assay. Combined use of the thionucleotide transcription reaction with the T1 nuclease assay allowed precise localization of the transcription start sites. The majority of MMTV RNA chains were initiated with guanosine 5'-O-(2-thiotriphosphate) at a template site 133 nucleotides upstream from a PvuII site that coincides with the right end of the long terminal repeat. However, some RNA chains were also initiated with adenosine 5'-O-(2-thiotriphosphate) and uridine 5'-O-(2-thiotriphosphate) at template sites within three nucleotides of the primary guanosine start site. When Mn2+ was substituted for Mg2+ in the transcription reaction, MMTV RNA chains were initiated with approximately the same efficiency, but the start site was shifted to a position approximately 40 nucleotides downstream from the physiological start site; in the presence of Mn2+, MMTV RNA chains were initiated only with guanosine 5'-O-(2-thiotriphosphate). When the nuclei were exposed to both Mn2+ and Mg2+, transcription initiated at the manganese-dependent site. Mn2+ also caused the transcription start site for 45 S pre-rRNA to shift about 10 nucleotides upstream from the physiologically correct start site.

Animals↗
PubMed1988

DNA and RNA sequence determination based on phosphorothioate chemistry.

The difference in reactivity between phosphate and phosphorothioate diesters is the basis of a chemical degradation scheme for the sequencing of DNA and RNA. The phosphorothioate groups are incorporated into the nucleic acid in four separate enzymatic reactions, with three of the natural nucleoside triphosphates and one alpha-thiotriphosphate in each reaction. Selective strand cleavage is achieved through alkylation to form the hydrolytically labile phosphorothioate triester. As an example, the sequence analysis is presented of M13 phage DNA and of RNA prepared by transcription with SP6 RNA polymerase.

Base Sequence↗
PubMed1988

5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis.

The application of T7 and lambda exonuclease to phosphorothioate-based oligonucleotide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (-)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or lambda exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.

Base Composition↗
PubMed1988

Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence of ethidium bromide.

A method for achieving strand specific nicking of DNA has been developed. Phosphorothioate groups were incorporated enzymatically into the (-)strand of M13 RF IV DNA. When such DNA is reacted with restriction endonucleases in the presence of ethidium bromide nicked DNA (RF II) is produced. All of the restriction enzymes tested linearised phosphorothioate-containing DNA in the absence of this dye. The strand specificity of the reaction was investigated by employing the ethidium bromide mediated nicking reaction in the phosphorothioate-based oligonucleotide-directed mutagenesis method. The mutational efficiencies obtained were in the region of 64-89%, indicating that these restriction enzymes hydrolyse the phosphodiester bond at the cleavage site of the unsubstituted (+)strand.

Base Composition↗
PubMed1988

Enhanced in vitro inhibition of HIV-1 replication by 3'-fluoro-3'-deoxythymidine compared to several other nucleoside analogs.

Nucleoside analogs were synthesized and tested for anti-HIV-1 activity in two in vitro test systems using MT-4 cells and peripheral blood lymphocytes. Cytotoxicity was assessed in both assays and additionally in several human lymphoblastoid cell lines. One analog, 3'-fluoro-3'-deoxythymidine, exhibited higher antiviral potency and slightly higher cytotoxicity than 3'-azido-3'-deoxythymidine. Analogs showing lower cytotoxicity than 3'-azido-3'-deoxythymidine demonstrated reduced antiviral activity. 3'-fluoro-3'-deoxythymidine may become an alternative chemotherapeutic agent because of its high antiretroviral potency. Its toxicity, however, deserves further investigation.

Cell Line↗
PubMed1988

[Bone structure changes in hip joint endoprosthesis implantation over the course of many years. A quantitative study].

The stability of implanted artificial joints is limited. One main factor for this is the change of the bone tissue near the implant. Therefore we studied femura with stem endoprostheses derived from autopsy. All specimens were cut in horizontal and longitudinal sections. X-rays were made of all sections. Then the specimens were embedded undecalcified and ground to 10 microns. The wellknown phenomenons at the bone cement border were found too. Direct bone cement contact is about 5%. The 4 studied femura show a higher porosity of cortical bone as controls. The loss of cortical bone is up to 40% after 55 months. There is a correlation between new load situation and the localisation of bone loss.

Aged↗