PubMed Health⌕ Search

Biomedical subjects

C C Kuenzle

Publications and source records attributed to C C Kuenzle.

At least 37 records · Page 2Linked to original sources
PubMed1981

An enzymatic method for microdetermination of aphidicolin: a promising anticancer drug.

We have developed a method, based on the in vitro inhibition of purified human DNA polymerase alpha, the major enzyme of DNA replication, which allows the rapid and accurate determination of pmol amounts of aphidicolin, a promising anticancer drug. The efficacy of this simple method was verified by the determination of aphidicolin in the liver, spleen, blood and urine of mice treated parenterically with the drug. Given its sensitivity and the avoidance of radioactive tracers, this enzymatic method is suitable for the determination of the drug in body fluids and tissue biopsies from living humans. It allows the detection and quantitation of aphidicolin in the presence of inactive metabolite(s) with very similar chemical structure(s) such as those generated by liver microsomal oxidases. The technique will also be useful to monitor the purification of the drug from cultures of Cephalosporium aphidicola.

Animals↗
PubMed1979

Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

By equilibrium dialysis a disadenosine 5',5'''-P1,P2-tetraphosphate (Ap4A) binding activity is shown to be present in mammalian cells. The Ap4A binding activity copurifies with DNA polymerase alpha during the isolation procedure, which includes chromatography on phospho-, DEAE-, and DNA-cellulose; gel filtration; sucrose gradient centrifugation; and electrophoresis in nondenaturing polyacrylamide gels. After these purification steps, DNA polymerase alpha appears to be homogeneous in nondenaturing polyacrylamide gels. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of such a purified DNA polymerase alpha preparation reveals seven distinct protein bands with apparent Mrs of 64,000, 63,000, 62,000, 60,000, 57,000, 55,000, and 52,000. By affinity labeling, the protein with Mr 57,000 has been shown to be the Ap4A-binding constituent of DNA polymerase alpha. The binding activity of DNA polymerase alpha for Ap4A is highly specific because neither structural analogs nor several other adenine nucleotides compete effectively with Ap4A for its binding site. The Ap4A binding site is lost in neuronal cells during maturation of rat brains concomitantly with the loss of DNA polymerase alpha and mitotic activity in those cells. From these results, DNA polymerase seems to be the intracellular target of Ap4A. This is discussed in respect to the recently reported of Ap4A to trigger DNA replication in quiescent mammalian cells [Grummt, F. (1978) Proc. Natl. Acad. Sci. USA 75, 371-375].

Adenine Nucleotides↗
PubMed1979

Functional roles of DNA polymerases beta and gamma.

The physiological functions of DNA polymerases (deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) beta and gamma were investigated by using neuronal nuclei and synaptosomes isolated from rat brain. UV irradiation of neuronal nuclei from 60-day-old rats resulted in a 7- to 10-fold stimulation of DNA repair synthesis attributable to DNA polymerase beta which, at this developmental stage, is virtually the only DNA polymerase present in the nuclei. No repair synthesis could be elicited by treating the nuclei with N-methyl-N-nitrosourea, but this way probably due to the inability of brain tissues to excise alkylated bases from DNA. The role of DNA polymerase gamma was studied in synaptosomes by using a system mimicking in vivo mitochondrial DNA synthesis. By showing that, under these conditions, DNA replication occurs in mitochondria, and exploiting the fact that DNA polymerase gama is the only DNA polymerase present in mitochondria, evidence was obtained for a role of DNA polymerase gamma in mitochondrial DNA replication. Based on these results and on the wealth of literature on DNA polymerase alpha, we conclude that DNA polymerase alpha is mainly responsible for DNA replication in nuclei, DNA polymerase beta is involved in nuclear DNA repair, and DNA polymerase gamma is the mitochondrial replicating enzyme. However, minor roles for DNA polymerase alpha in DNA repair or for DNA polymerase beta in DNA replication cannot be excluded.

Animals↗
PubMed1979

Fetal and adult albumins are indistinguishable by immunological and physicochemical criteria.

The existence of a functionally immature fetal albumin has been postulated to explain the reduced ability of newborn plasma to bind bilirubin and various drugs. In support of this, cord and adult albumin, isolated by a simple salting-out technique, were reported to differ in electrophoretic and chromatographic properties and in their resistance to alkali and proteolytic enzymes. However, the interpretation of these findings has since been questioned. To resolve this controversy, we have purified to homogeneity human serum albumins from pooled umbilical cord and adult donor plasma. The two albumins were compared and found to be indistinguishable by polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, as well as by immunoelectrophoresis and double immunodiffusion using specific antibodies against both albumins. Furthermore, the amino acid compositions, the aminoterminal sequence (Asp-Ala-His-Lys-Ser-Glu-Val-Ala-), the carboxy terminus (Leu), and the peptide fingerprints were identical in the two albumins. No significant differences were found by circular dichroism in the ultraviolet (200-350 nm). Binding studies with bilirubin showed association constants of 3.7 +/- 0.7 x 10(7) M(-1) for cord and 2.9 +/- 0.3 x 10(7) M(-1) for adult albumin, respectively. The circular dichroic spectra of 1:1 bilirubin.albumin complexes showed considerable variation between the batches but were not significantly different. The only difference was found in the fluorescence spectra of the bilirubin.albumin complexes, in which complexes with adult albumin showed only 75% of the relative fluorescence exhibited with cord albumin. The combined results nevertheless strongly indicate that fetal and adult albumins are very similar, if not identical.

Amino Acid Sequence↗
PubMed1979

High C + G content in parapoxvirus DNA.

The DNAs of eight parapoxviruses (four stomatitis papulosa viruses isolated from infected calves, a pseudocowpox virus isolated from a teat lesion of an infected cow and three orf viruses, one isolated from an infected sheep and two isolated from human infections) were analysed in CsCl gradients. The mole % of G+C was calculated from the buoyant density and found to be approx. 63% for all virus isolates examined. Parapoxvirus DNA thus has by far the highest G+C content of all poxvirus DNAs so far examined.

Animals↗
PubMed1979

DNA polymerase beta from brain neurons is a repair enzyme.

DNA polymerase beta was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian beta-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no alpha-polymerase, 99.2% beta-polymerase and only 0.8% gamma-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since beta-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the beta enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2',3'-dideoxyribosylthymine 5'-triphosphate (d2TTP), a potent beta-polymerase inhibitor. However, the presence of minute amounts of gamma-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase gamma in DNA repair. In view of the similarity of the neuronal DNA polymerase beta with all other mammalian beta-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all beta-polymerases.

Animals↗
PubMed1977

Identity of DNA polymerase gamma from synaptosomal mitochondria and rat-brain nuclei.

DNA polymerase gamma and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase gamma and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems; essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements of Mn2+ (0.1 mM), Mg2+ (5 mM) and pH (8.0); template preferences, poly(A) - (dT)20-25 larger than activated DNA larger than poly(dA) - (dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA; molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pI 5.4). We therefore conclude that brain nuclear DNA polymerase gamma and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical.

Animals↗
PubMed1977

Variation of DNA polymerases-alpha, -beta. and -gamma during perinatal tissue growth and differentiation.

The activities of the three known DNA polymerases-alpha, beta-, and -gamma were determined in rat brain neurons, cardiac muscle and spleen, and were correlated with the rate of cell proliferation during perinatal development. In neurons and cardiac muscle, which stop dividing before birth, DNA polymerase-alpha activity drops sharply from a high level with the approach of term and disappears at approximately two weeks postnatal age. In contrast, alpha-polymerase activity is almost absent in spleen during late gestation, when the rate of cell division is low, and increases abruptly after birth with the sudden onset of cell proliferation. These data give further evidence for an involvement of DNA polymerase-alpha in DNA replication. DNA polymerase-beta and -gamma activities show essentially no correlation with the rate of cell division. Thus, these enzymes are probably responsible for repair type processes rather than for DNA replecation.

Aging↗