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D E Kizer

Publications and source records attributed to D E Kizer.

At least 19 recordsLinked to original sources
PubMed1987

Evidence for nucleoside channeling in vivo: deoxythymidine incorporation into rat liver dTTP and nuclear matrix DNA.

Previous studies in prokaryotes and in eukaryotic cell lines have indicated the possible existence of more than one dTTP pool accessible to DNA synthesis. To investigate this possibility in eukaryotes in vivo, the incorporation of [3H] deoxythymidine into nuclear matrix-attached DNA and intracellular dTTP was examined in regenerating rat liver. The labeling of matrix DNA reached a maximum after a 5 min pulse and then began to rapidly decrease. Conversely, [3H] deoxythymidine incorporation into dTTP began to increase after 5 min and peaked 10 min after injection. Since the peak specific activity for [3H] deoxythymidine incorporation into matrix DNA precedes that into dTTP, there seems to be channeling of exogenous thymidine directly to sites of DNA replication, bypassing existing nucleotide pools.

Animals↗
PubMed1987

Assessment of salvage pathways utilized for incorporation of exogenous pyrimidine nucleosides into DNA of guinea pig lymphocytes stimulated by Con A.

The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.

Amides↗
PubMed1985

Distribution patterns for 5-methylcytosine among apurinic DNAs from several sources.

Purified DNA from the liver of rats, mice, rabbits, and guinea pigs, from guinea pig lymph nodes, from hyperplastic nodules induced in rat liver by feeding with 2-(acetylamino)fluorene, and from Escherichia coli cells was made apurinic by reaction with diphenylamine. After chromatographic separation of pyrimidine tracts (isostichs or isoplyths) according to the number of contiguous pyrimidines, semilog plots of tract frequency vs. the number of contiguous pyrimidines were linear, plots for DNA from several sources differed from one another, and all deviated significantly from randomness. Similar semilog plots for coding sequences among 60 mammalian genomes or 28 rat tissue genomes were intermediate among slopes for isolated DNA. Individual isostichs were hydrolyzed, and their constituent pyrimidine bases were analyzed by high-pressure liquid chromatography. Among isostichs from isolated DNAs, the distribution of Thy and Cyt contents differed markedly from the distribution of 5-methylcytosine (5-Me-Cyt); e.g., although isostich 1 contained 45-49% of 5-Me-Cyt, amounts of Thy or Cyt did not exceed 25%. Semilog plots of normalized values for tract frequency or the content of 5-Me-Cyt vs. isostich number were essentially superimposable; thus, among the first five pyrimidine tracts of a particular tissue or E. coli DNA, the number of tracts per 5-Me-Cyt moiety was essentially constant. The data showed that 5-Me-Cyt and/or dCyd-dGuo dinucleotides have a distribution throughout DNA structure that superimposes the distribution of pyrimidine tract frequency and suggests that regulatory 5-Me-Cyt moieties are principally located at 3' termini of pyrimidine tracts.

5-Methylcytosine↗
PubMed1985

Assessment of rat liver microsomal epoxide hydrolase as a marker of hepatocarcinogenesis.

The influence of eleven xenobiotics on the activity and amount of hepatic microsomal epoxide hydrolase was determined. Activity was assayed using three different substrates after rats were fed, throughout 3 weeks, diets containing one of six hepatocarcinogens, viz. 2-acetylaminofluorene, 3'-methyl-4-dimethylaminoazobenzene, 4'-fluoro-4-dimethylaminoazobenzene, thioacetamide, aflatoxin B1 and ethionine. Five hepatocarcinogens induced activity 4- to 10-fold; ethionine was relatively ineffective as an inducer. Two non-carcinogenic analogues of hepatocarcinogens, viz. fluorene and p-aminoazobenzene, caused no appreciable increase in enzyme activity, but phenobarbital, barbital and 1-naphthylisothiocyanate induced activity 2- to 3-fold. All eleven xenobiotics increased the amount of microsomal epoxide hydrolase 2- to 9-fold when examined immunochemically using either a radial diffusion assay or an enzyme-linked immunosorbent assay (ELISA). Serum glutamic oxaloacetic acid transaminase activity was not appreciably elevated by feeding ten of the xenobiotics, suggesting that inductions were not owing to toxicity. Using ELISA, microsomal epoxide hydrolase was detected in post-microsomal (PM) supernatant fractions from control rat liver, thus confirming an earlier report by Gill et al. [Carcinogenesis 3, 1307 (1982)]. The eleven xenobiotics induced the amount of ELISA-detectable antigen in PM supernatant fractions by 3- to 34-fold. Longer centrifugation of PM supernatant fractions yielded a pellet fraction that contained 92 +/- 1.2% of the ELISA-detectable antigen irrespective of the xenobiotic regimen. Relationships between xenobiotic induction of microsomal epoxide hydrolase activity and amount and hepatocarcinogenesis are discussed.

Animals↗
PubMed1985

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA.

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.

DNA↗
PubMed1983

Asymmetric distribution of exogenous thymidine among pyrimidine isostichs suggests compartmentalization of replicative DNA synthesis during regeneration in rat liver.

The distribution of radioactivity among pyrimidine isostichs (or isoplyths) of DNA from 24-h regenerating rat liver was studied with [3H]Thd, [14C]orotate or with inorganic 32Pi. Expression of incorporated radioactivity as log10% of total radioactivity recovered for each of the 11 pyrimidine isostichs detected showed that radioactivity from [3H]Thd was asymmetrically distributed among the isostichs, i.e., 3H radioactivity failed to access regions of DNA yielding lower molecular weight pyrimidine isostichs as efficiently as it accessed regions yielding higher molecular weight pyrimidine isostichs. The thymine (T) content of isostichs exceeded that of cytosine (C), i.e., T/C ratios for the first 10 isostichs averaged 1.43 +/- 0.08 and 1.28 +/- 0.05, depending on the method of analysis; furthermore, the T/C ratio for isostich 1 was significantly higher than ratios for isostichs 2 through 10. Asymmetric distributions of [3H]Thd radioactivity also were seen at 18 or 30 h post-partial hepatectomy. Thus, radioactivity from [3H]Thd, a DNA precursor from the salvage pathway, failed to efficiently access lower molecular weight isostichs despite thymine enrichment, suggesting that thymine moieties were supplied from additional sources. Radioactivity from [14C]orotate accessed lower molecular weight pyrimidine tracts more efficiently than [3H]Thd, but less efficiently than it accessed higher molecular weight isostichs, resulting in an asymmetric distribution of 14C radioactivity. This result suggested that appreciable quantities of thymine and cytosine moieties utilized for DNA synthesis were supplied de novo, but other sources also were utilized. Radioactivity from 32Pi, a de novo precursor, was distributed symmetrically, i.e., the slope among lower molecular weight isostichs increased enough that it was indistinguishable from slopes for intermediate and higher molecular weight isostichs. Since 32P radioactivity among lower molecular weight isostichs reflects appreciable contributions of de novo phosphate moieties from both pyrimidine- and purine-containing deoxynucleoside triphosphates, opportunities for observing contributions of 32P radioactivity from pathways other than the de novo pathways appeared to lie beyond limits of detectability. The distribution of radioactivity from labeled DNA precursors among lower molecular weight pyrimidine tracts (a) indicate that thymine moieties are contributed by both salvage and de novo pathways; (b) support the possibility that cytosine moieties also are contributed by both pathways; and (c) support the 'replitase' concept for channeling dNTPs to replicating forks.

Animals↗
PubMed1982

Evidence that free polysomes are not the precursors of membrane-bound polysomes in rat liver.

We tested, in rat liver, the postulate that free polysomes were precursors of membrane-bound polysomes. Three methods were used to isolate free and membrane-bound ribosomes from either post-nuclear or post-mitochondrial supernatants of rat liver. Isolation and quantitation of 28 S and 18 S rRNA allowed determination of the 40 S and 60 S subunit composition of free and membrane-bound ribosomal populations, while pulse labeling of 28 S and 18 S rRNA with [6-14C) orotic acid and inorganic (32P] phosphate allowed assessment of relative rates of subunit renewal. Throughout the extra-nuclear compartment, 40 S and 60 S subunits were present in essentially equal numbers, but, free ribosomes contained a stoichiometric excess of 40 S subunits, while membrane-bound ribosomes contained a complementary excess of 60 S subunits. Experiments with labeled precursors showed that throughout the extra-nuclear compartment, 40 S and 60 S subunits accumulated isotopes at essentially equal rates, however, free ribosomes accumulated isotopes faster than membrane-bound ribosomes. Among free ribosomes or polysomes, 40 S subunits accumulated isotopes faster than 60 S subunits, but, this relationship was not seen among membrane-bound ribosomes. Here, 40 S subunits accumulated isotope more slowly than 60 S subunits. This distribution of labeled precursors does not support the postulate that free polysomes are precursors of membrane-bound polysomes, but, these data suggest that membrane-bound polysomes could be precursors of free polysomes.

Animals↗
PubMed1981

Differences in the distribution of phosphate content in the ribosomal subunit proteins of free and membrane-bound ribosomes from normal and regenerating rat liver.

Proteins of membrane-bound ribosomes from normal liver contained 60-70% more phosphate than did proteins from free ribosomes. This difference was not a reflection of the phosphate contents of respective 40 S subunits. Instead, it was owing to the presence of high levels of phosphorylated proteins in the 60 S subunits, i.e., phosphate contents equal to or greater than those for 40 S subunits. The proteins of membrane-bound 60 S subunits contained twice the phosphate as free 60 S subunits. In regenerating rat liver, membrane-bound ribosomes had increased phosphate in the proteins of the 40 S subunits and decreased phosphate in proteins of the 60 S subunit when compared to controls for normal rat liver. No significant changes occurred in the proteins of free ribosomes from regenerating rat liver. These findings are discussed with respect to (a) the importance of assessing total phosphate contents of proteins in the study of ribosomal protein phosphorylation, and (b) the possible involvement of ribosomal protein phosphorylation in the segregation of ribosomes into free and membrane-bound populations and the regulation of these distributions to meet changes in the translational demands of the cell.

Animals↗
PubMed1978

Identification of epoxide hydrase as the preneoplastic antigen in rat liver hyperplastic nodules.

A liver microsomal protein, previously referred to as preneoplastic antigen, from hyperplastic nodules of rats fed a diet containing 2-acetylaminofluorene has been identified as the enzyme epoxide hydrase [glycol hydro-lyase (epoxideforming), EC 4.2.1.63]. Purified preneoplastic antigen from hyperplastic nodules and purified rat liver microsomal epoxide hydrase are immunochemically identical on the basis of Ouchterlony double-diffusion analysis. In addition, the purified proteins have identical minimum molecular weights in sodium dodecyl sulfate/polyacrylamide gels, and both proteins catalyze the hydration of arene oxides to dihydrodiols. Chronic feeding of 2-acetylaminofluorene to rats results in a 5- to 7-fold increase in epoxide hydrase activity in rat liver. The induced level of the enzyme is maintained in developing hyperplastic nodules and hepatomas but not in the nontumor tissue after removal of the carcinogen from the diet.

2-Acetylaminofluorene↗
PubMed1978

Purification and quantitation of preneoplastic antigen from hyperplastic nodules and normal liver.

The protein in microsomal membranes of hyperplastic nodules of livers from rats fed a diet containing 2-acetyl-aminofluorene referred to as the preneoplastic antigen has been purified to a nearly homogeneous state. A protein sharing identical immunodeterminants based on double agarose diffusion and immunoelectrophoresis has also been detected in deoxycholate-solubilized microsomes from normal liver, but it cannot be detected by gel immunodiffusion in the absence of detergent. There is approximately 4 times as much of this antigen in hyperplastic nodule microsomes as there is in normal microsomes as determined by electroimmunoassay. The antigen from nodules is not more than one-half the molecular weight of the antigen derived from normal microsomes in 0.2% deoxycholate. These findings are consistent with the hypothesis that an altered state of membranes exists in hyperplastic nodule endoplasmic reticulum, resulting in increased amounts of immunoreactive preneoplastic antigen. The nodule protein has a decreased association with its membrane matrix.

2-Acetylaminofluorene↗
PubMed1975

Terbium binding to ribosomes and ribosomal RNA.

Terbium binding to rat liver ribosomes and ribosomal RNA (rRNA) was examined by equilibrium dialysis and fluorescence spectroscopy. Upon binding to ribosomes and rRNA, the enhancement of terbium fluorescence emission at both 488 and 541 nm was dependent only upon the amount of bound terbium and independent of ionic strength. Binding profiles for ribosomes and rRNA suggested that terbium was bound to ribosomes primarily through rRNA interactions. Data suggested that terbium mimicked characteristics previously described for interactions between ribosomes and magnesium. It is proposed, therefore, that fluorescence of terbium bound to ribosomes may prove useful in studies on the nature and extent of interactions between ribosomes and magnesium.

Animals↗
PubMed1975

Does phospholipase C stimulate thymide kinase activity of rat liver extracts prepared after partial hepatectomy.

Our purpose was to determine whether phospholipase C stimulated thymidine kinase activity of regenerating rat liver. We determined effects of phospholipase C upon TMP formation by rat liver extracts prepared at 0, 12, 24, 36 and 48 hr following partial hepatectomy. Data were obtained which supported these conclusions: (a) Commercial preparations of phospholipase C contained nucleoside phosphotransferase activity; (b) phospholipase C exerted no appreciable stimulatory influence upon thymidine kinase activity of regenerating rat liver; and (c), apparent stimulation of thymidine kinase was associated with linked activities of two enzymes, viz., liver extract-ATPase activity and nucleoside phosphotransferase activity.

Adenosine Monophosphate↗