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2.
Genetic and biochemical evidence suggests there are at least three DNA polymerases required for replication in eukaryotic cells. However, Drosophila embryonic cells have a very short duration S phase which is regulated differently. To address the question of whether embryos utilize different DNA polymerases, we employed Mono Q anion exchange chromatography to resolve the DNA polymerase activities. Two types of DNA polymerase, DNA polymerase delta and DNA polymerase alpha, were distinguished by: 1. copurification of DNA primase or 3'-5'exonuclease activities; 2. immunoblot analysis with alpha-specific polyclonal antisera; 3. sensitivity to aphidicolin and BuPdGTP; and 4. processivity measurements with and without Proliferating Cell Nuclear Antigen. These observations suggest that Drosophila embryos, similar to nonembryonic cells, have both alpha- and delta-type DNA polymerases. 相似文献
3.
Polyamine-mediated turnover of ornithine decarboxylase in Chinese-hamster ovary cells. 总被引:3,自引:1,他引:2
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The major secreted isoenzyme of human prostatic acid phosphatase (PAcP) (EC 3.1.3.2), which catalyses p-nitrophenyl phosphate (PNPP) hydrolysis at acid pH values, was found to have phosphotyrosyl protein phosphatase activity since it dephosphorylated three different phosphotyrosine-containing protein substrates. Several lines of evidence are presented to show that the phosphotyrosyl phosphatase and PAcP are the same enzyme. A highly purified PAcP enzyme preparation which contains a single N-terminal peptide sequence was used to test for the phosphotyrosyl phosphatase activity. Both activities comigrated during gel filtration by high performance liquid chromatography. Phosphotyrosyl phosphatase activity and PNPP acid phosphatase activity exhibited similar sensitivities to different effectors. Both phosphatase activities showed the same thermal stability. Specific anti-PAcP antibody reacted to the same extent with both phosphatase activities. PNPP acid phosphatase activity was competitively inhibited by the phosphotyrosyl phosphatase substrate. To characterize further the phosphotyrosyl phosphatase activity, the Km values using different phosphoprotein substrates were determined. The apparent Km values for phosphorylated angiotensin II, anti-pp60src immunoglobulin G and casein were in the nM range for phosphotyrosine residues, which was about 50-fold lower than the Km for phosphoserine residues in casein. 相似文献
4.
Nuclear and cytoplasmic protein kinases were measured during the traverse of synchronous CHO cultures through G1 into S phase. Cells were synchronized by selective detachment of cells blocked in metaphase using colcemid. Nuclei were isolated and the protein kinases extracted from the nuclear preparation with 0.6 M NaCl. This procedure solubilized greater than 90% of the total protein kinase activity present in the nuclear preparation. DEAE chromatography of this extract showed 5 apparently different ionic forms of nuclear protein kinases. The nuclear protein kinases preferred casein and phosvitin to histone as substrates and were cyclic AMP-independent. Nuclear protein kinase activities increased greater than two-fold, when expressed as units of activity per cell nucleus, during G1 phase traverse, concomitant with a 70% increase in nuclear non-histone proteins (those soluble in 0.6 M NaCl). This resulted in only a 40% increase in the specific activities (units/microgram protein in 0.6 M NaCl extractable nuclear fraction) of these enzymes as cells progressed through G1 into S phase. This was in contrast to cytoplasmic cyclic AMP-dependent protein kinase activities which also increased two-fold during progression through G1 phase while total cellular protein increased less than 20%. Activation of, as well as synthesis of, cyclic AMP-dependent cytoplasmic protein kinases during G1 phase suggests a regulatory mechanism for precise temporal phosphorylation, whereas the constant specific activity in nuclear kinases during cell cycle is more compatible with the maintenance of bulk phosphorylation processes in the nucleus. 相似文献
5.
Phosphorylation of nuclear and DNA-binding proteins in proliferating and quiescent mammalian cells.
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The dependence of cell proliferation on nuclear protein phosphorylation was studied with exponential-phase and stationary-phase cultures of Chinese-hamster ovary cells. Nuclear proteins were fractionated, according to their DNA-binding affinities, by using sequential extractions of isolated nuclei with increasing concentrations of NaCl. When viable whole cells were labelled with H332PO4, phosphorylation of nuclear proteins was found to be lower in quiescent cells than in proliferating cells. Phosphorylation of nuclear proteins soluble in 0.30M-NaCl (less than 50% of these proteins bind to DNA) was greater than for those proteins soluble in higher salt concentrations (80-100% of these proteins bind to DNA). Cyclic AMP enhanced the phosphorylation of nuclear proteins soluble in 0.3 m-NaCl by 40-50%, and this stimulation was independent of cell growth. Cyclic AMP also increased the phosphorylation of nuclear proteins soluble in 0.6M-NaCl and 2.0M-NaCl by 40-50% in exponential-phase cultures, but not in stationary-phase cultures. Several examples of specific phosphorylation in response to cyclic AMP were observed, including a 35000-mol.wt. protein in the 0.30 M-NaCl-soluble fraction and several proteins larger than 100000 molecular weight within this fraction. A major peptide of molecular weight approx. 31000 extracted with 0.6M-NaCl was also phosphorylated. Its phosphorylation was independent of cyclic AMP in exponential-phase cultures, and it was not phosphorylated in plateau-phase cells. These changes in cell-growth-dependent phosphorylation occurred in the absence of any apparent qualitative changes in the nuclear protein molecular-weight distributions. These data demonstrate that (1) phosphorylation of nuclear proteins is dependent on the culture's proliferative status, (2) both cyclic AMP-dependent and cyclic AMP-independent specific phosphorylation occurs, and (3) the cyclic AMP-dependent growth-independent phosphorylation that occurs does not appear to be a modification of DNA-binding proteins, whereas the cyclic AMP-dependent growth-dependent phosphorylation does involve modification of DNA binding proteins. 相似文献
6.
Chinese hamster ovary cells, selected in mitosis and plated into medium containing hydroxyurea, can progress through G1 and enter S phase although bulk DNA synthesis is prevented. As the cells progress through G1 in the presence of hydroxyurea, ornithine decarboxylase activity remains low while general protein synthesis appears unaffected. After hydroxyurea is removed, ornithine decarboxylase activity increases, but only after approximately 20% of the DNA has been replicated. These results suggest that ornithine decarboxylase induction is not essential for cellular progression into S phase but is required for the completion of DNA synthesis. 相似文献
7.
Synthesis and uptake are two important regulated mechanisms by which eukaryotic cells maintain polyamine levels. The role that loss of synthesis and/or uptake regulation plays in mediating putrescine toxicity was investigated by comparing toxicity in an ornithine decarboxylase (ODC)-deficient Chinese hamster ovary cell line (C55.7) with a functional putrescine transport system and an ODC-overproducing rat hepatoma cell line (DH23b), which are transport regulation deficient. When C55.7 cells were transfected with either mouse ODC (M) or trypanosome ODC (Tb), intracellular putrescine content increased slightly in C55.7(Tb-ODC), compared to C55.7(M-ODC), due to the lack of response of Tb-ODC to polyamine regulation. The increase in putrescine content resulting from loss of ODC regulation had no impact on cell growth and viability. When the feedback repression of polyamine uptake was blocked with cycloheximide, C55.7 cells transfected with either ODC construct accumulated very high levels of putrescine from the medium, and underwent apoptosis in a putrescine dose-dependent manner. A similar correlation of deregulated putrescine uptake and increased apoptotic cells was observed in DH23b cells. These data demonstrate that loss of feedback regulation on the polyamine transport system, but not ODC activity, is sufficient to induce apoptosis. Thus, downregulation of the transport system is necessary to prevent accumulation of cytotoxic putrescine levels in rodent cells. 相似文献
8.
Kai Hilpert Dirk FH Winkler Robert EW Hancock 《Biotechnology & genetic engineering reviews》2013,29(1):31-106
Spatial organization of metabolic enzymes may represent a general cellular mechanism to regulate metabolic flux. One recent example of this type of cellular phenomenon is the purinosome, a newly discovered multi-enzyme metabolic assembly that includes all of the enzymes within the de novo purine biosynthetic pathway. Our understanding of the components and regulation of purinosomes has significantly grown in recent years. This paper reviews the purine de novo biosynthesis pathway and its regulation, and presents the evidence supporting the purinosome assembly and disassembly processes under the control of G-protein-coupled receptor (GPCR) signaling. This paper also discusses the implications of purinosome and GPCR regulation in drug discovery. 相似文献
9.
To assess the suitability of the use of structural alerts to identify the skin-sensitising properties of chemicals, the 40 originally published structural alerts for the prediction of skin-sensitisation properties used by the DEREK system (which now contains 59 structural alerts), have been evaluated against a database developed in the German Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), which contains data submitted under the procedure for notifying new chemicals within the European Union. The evaluation of the 40 structural alerts used in DEREK revealed that eight of the 40 structural alerts for the prediction of skin-sensitising potential could be used without any further refinement. Ten structural alerts may need further specifications or refinements in order avoid false-positive predictions - proposals for refinement are discussed. Not enough substances were found within the BgVV database (containing data for more than 1000 substances) to evaluate ten of the DEREK substructures; hence, for these structural alerts, a judgement on their suitability for prediction of skin-sensitisation properties in expert systems is not possible. For 12 structural alerts, no comparative result could be obtained, because these rules did not "fire" for any of the examined chemicals. As a general result of the evaluation process, the approach of using structural alerts for the prediction of skin-sensitising properties of chemicals proved to be reliable. Proposals are given for a refinement of the structural alerts for prediction of contact allergy used in the DEREK system. In addition, advice and several preconditions are given, that apply when trying to teach a computer system to use structural alerts to predict toxicological properties. 相似文献
10.
Gerner I Zinke S Graetschel G Schlede E 《Alternatives to laboratory animals : ATLA》2000,28(5):665-698
The notification procedure of the European Union (EU) for new chemicals requires the application of protocols on physicochemical and toxicological tests for the evaluation of physicochemical properties and probable toxic effects of each notified substance. A computerised database was developed from data sets and toxicological test protocols relating to substance properties responsible for skin and eye irritation/corrosion. To develop specific structure-activity relationship (SAR) models and to find rules for a decision support system (DSS) to predict local irritation/corrosion, physical property data, chemical structure data and toxicological data for approximately 1300 chemicals, each having a purity of 95% or more, were evaluated. The evaluation demonstrated that the lipid solubility and aqueous solubility of a chemical are relevant to, or - in some cases - responsible for, the observed local effects of a substance on the skins and eyes of rabbits. The octanol/water partition coefficient and the measured value of the surface tension of a saturated aqueous solution of the substance give additional information that permits the definition of detailed SAR algorithms that use measured solubility values. Data on melting points and vapour pressure can be used to assess the intensity and duration of local contact with a chemical. Considerations relating to the reactivity of a pure chemical can be based on molecular weight and the nature of the heteroatoms present. With respect to local lesions produced following contact with the skin and eyes of rabbits, the data evaluation revealed that no general "local irritation/corrosion potential" of a chemical can be defined. A variety of mechanisms are responsible for the formation of local lesions on the skin or in the eyes: serious lesions are produced by mechanisms different from those that cause moderate irritation in these organs. In order to develop a DSS that uses the information extracted from the database, chemical main groups were categorised on the basis of their empirical formulae, and rules were defined of the type IF (physicochemical property) A, THEN not (toxic) effect B, based on correlations between specific local effects and measured physicochemical values. Other rules of the type IF substructure A, THEN effect B were developed based on correlations between specific local effects and the submitted structural formulae. Reactive chemical substructures relevant to the formation of local lesions and rules for the prediction of the absence of any skin irritation potential were identified. Proposals are made relating to the development of alternatives to eye irritation testing with rabbits. 相似文献