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P Reinhard

Publications and source records attributed to P Reinhard.

36 records · Page 2Linked to original sources
PubMed1988

Lee et al. reply.

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Journal Article↗
PubMed1988

DNA repair synthesis in human fibroblasts requires DNA polymerase delta.

When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II (Crute, J. J., Wahl, A. F., and Bambara, R. A. (1986) Biochemistry 25, 26-36). Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone. This result suggests that cytosol-depleted permeabilized DNA repair-defective human fibroblasts and HeLa DNA polymerase delta might be exploited to provide a functional assay for purifying active DNA repair factors from DNA repair-proficient cells without a preknowledge of their function.

DNA Polymerase II↗
PubMed1983

Effect of a putative antidepressant, Rolipram, on the circadian running wheel activity of a day-active chipmunk, Eutamias sibiricus.

Siberian chipmunks (Eutamias sibiricus) were kept individually in small cages attached to a running wheel. Under continuous illumination of two different light intensities (0.4-0.9 lux and 400-1200 lux) the influence of Rolipram, a putative new antidepressant, on the period length tau was tested. Under both conditions Rolipram caused a lengthening of tau, a decrease of activity time alpha and an increase of rest time delta, resulting in a decrease of the alpha/delta ratio. These effects of Rolipram could be due to a slowing down of the circadian oscillatory system or an influence on the sensitivity towards light.

Animals↗
PubMed1979

An assay system for factors involved in mammalian DNA replication.

An assay for cellular factors stimulating DNA synthesis by partially lysed CHO cells is presented. The assay is based on the observation that in highly lysed cells, DNA synthesis, as determined by [3H]dTTP incorporation, was only 2-5% of that in gently lysed cells, and that this low level of DNA synthesis could be increased by a factor of approx. 50 by the addition of CHO cell extract (i.e. supernatant of a cell homogenate subjected to high-speed centrifugation). Highly lysed cells were obtained by treatment with 0.1% Brij-58 and 240 mM KCl, while for the preparation of gently lysed cells, 0.01% Brij-58 and 80 mM KCl were used. Incorporation of [3H]dTTP reflected DNA synthesis qualitatively similar to that in intact cells. It was semiconservative, and no repair synthesis was detected unless cells were irradiated with ultraviolet light prior to parital lysis. DNA molecules of 4 S were synthesized and converted to DNA of more than 25 S via 6-12-S intermediates. DNA synthesis was restricted to nuclei from cells in S phase, and cell extract did not induce DNA synthesis in nuclei from cells in G1 phase. Stimulation of DNA synthesis by cell extract was concentration-dependent. Cell extract activity was recovered to more than 50% after (NH4)2SO4 precipitation. Heat-inactivation experiments suggested that cell extract contained at least tow factors timulating DNA replication. This system may, therefore, be used for the purification and characterization of factors participating in DNA replication of mammalian cells.

Animals↗
PubMed1978

Synthesis of a mammalian parvovirus in Brij-58-lysed cells.

An in vitro system was developed, prepared by lysis of parvovirus LuIII-infected cells with Brij-58. In this subcellular system, DNA of viral genome size and synthesized and subsequently packaged into particles with physicochemical properties of mature LuIII virions.

Cell Line↗
PubMed1978

[The ACTH content of plasma in fetal and newborn swine].

ACTH concentration has been estimated radioimmunologically in fetal plasma (100th day of gestation) and in plasma of newborn piglets during the first 24 hours of life and in sows. In comparison to the values of ACTH in sows at the 100th day of gestation during anaesthesia (175 pg/ml) and sows at parturition (235 +/- 77 pg/ml) the concentration in fetal (558 +/- 163 pg/ml) and newborn piglets (448 +/- 158 pg/ml) was much higher. On an average ACTH concentration increased during the first 24 hours of life up to 998 +/- 628 pg/ml. The results are compared to those in other species.

Adrenocorticotropic Hormone↗
PubMed1977

Semiconservative DNA replication in vitro. I. Properties of two systems derived from mouse P-815 cells by permeabilization or lysis with Brij-58.

To study DNA replication in vitro, mouse P-815 cells were either permeabilized by hypotonic treatment or gently lysed with the detergent Brij-58. In the presence of KCl, EGTA, creatine phosphokinase, creatine phosphate, sucrose, dithiothreitol, CTP, GTP, UTP, and HEPES at pH 7.8, both in vitro systems required similar concentrations of all four deoxyribonucleoside triphosphates, ATP, Mg2+, and dextran. Incorporation of [3H] dTTP was due to semiconservative DNA replication and was restricted to S-phase nuclei. No repair replication was detectable. In the first 20 min, the rate of DNA replication in vitro was 30--40% of the in vivo rate, and after 60 min, about 3% of the genome were replicated. Preexisting bulk DNA was not fragmented during permeabilization or lysis with Brij-58 and during replication in vitro. Size distributions of growing strands in vitro were similar to those found in vivo. Neither cycloheximide (100 mug/ml) nor hydroxyurea (3 mM) inhibited DNA replication in vitro.

Adenosine Triphosphate↗
PubMed1977

Semiconservative DNA replication in vitro. II. Replicative intermediates of mouse P-815 cells.

DNA chain growing during semiconservative replication was studied using both in vitro systems described in the preceding paper (preceding paper, ref 1) 3H-Labeled, 4-S Okazaki fragments synthesized in vivo just prior to permeabilization or lysis with Brij-58 were metabolically stable and quantitatively chased into high molecular weight DNA (20--100 S) during a subsequent incubation in vitro. Thus, DNA replication continued in vitro at the same growing points that were active in vivo. After a 20-s pulse at 30 degress C in vitro, more than 50% of incorporated radioactivity was found in the 4 S region of alkaline sucrose gradients suggesting a totally discontinuous mode of DNA chain growth. If the pulse were followed by a 1-min chase, 4-S molecules were converted into 6--12-S intermediates which upon continued incubation were joined with growing 20--100-S molecules (replicon-sized chains). Formation of all three classes of replicative intermediates, Okazaki fragments, 6--12-S intermediates, and 20--100-S molecules, occurred in vitro at least during the first 20 min. During this time, average rates of DNA chain growth and overall DNA synthesis were reduced to about the same extent, if compared to rates of intact cells. Thus, reduced chain growth rates appear to reflect primary deficiences of our in vitro systems, while initiation of replicative intermediates still occurs.

Animals↗