Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo

In this article the structural analysis of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene (AAF) to rat liver DNA in vivo is described. This compound appears to result from the formation of a covalent bond between carbon-3 of the aromatic ring and the amino group of guanine. Experimental evidence from three different approaches has led to the identification of the structure of the persistently DNA-bound AAF moity. First, [3-³H, 9-¹⁴C]N-acetoxy-AAF was reacted with DNA in vitro. As reported previously, a minor product was isolated from enzymatic digests of the reacted DNA, which had chemical and chromatographic properties identical to those of the persistent—AAF moiety in DNA in vivo. The ration ³H/¹⁴C of this product had diminished to the same extent as 3-CH₃S-AAF resulting from the reaction of methionine with [3-³H, 9-¹⁴C]N-acetoxy-AAF.Secondly, reaction of [9-¹⁴C]N-acetoxy-AAF with DNA, which was tritiated in the C-8 positions of the purines, did not result in removal of tritium in the persistent fraction obtained after acid hydrolysis, thus excluding substitution at C-8 and N-7 of guanine. Finally, by reacting N-OSO₃-K-AAF with deoxyguanosine in dimethylsulfoxide-triethylamine, a compound could be isolated, which was identified as 3-(deoxyguanosin-N²-yl)-AAF based on its NMR spectrum and on the mass spectrum of the corresponding guanine derivative obtained after removing deoxyribose by acid hydrolysis. This compound appeared to be identical with the persistently bound form present in DNA hydrolysates from rat liver after injection of [2′-³H]N-hydroxy-AAF.     

Formation of electrophilic N-acetoxyarylamines in cytosols from rat mammary gland and other tissues by trans-acetylation from the carcinogen N-hydroxy-4-acetylaminobiphenyl

The 105 000 × g supernatant fractions of various rat tissues catalyze the transfer of the N-acetyl group of certain carcinogenic aromatic acethydroxamic acids to the O atom of aromatic hydroxylamines. The resulting N-acetoxyhydroxylamines are strongly electrophilic and have been detected and analyzed through their reaction with N-acetylmethionine to yield methylmercaptoaminoarenes.Of the rat tissues studied the liver had the highest activity; kidney and small intestinal mucosa were about 15–20% as active. The transacetylase activities of these tissues were similar with respect to their ability to use either N-hydroxy-2-acetylaminofluorene (N-hydroxy-AAF or N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP) as acetyl donors, their stability on storage at 2–3°C, and their elution patterns from Sephadex G-100 columns. Low transacetylase activity was found in spleen and muscle.Mammary tissue from 16–21 day pregnant rats had 20% of the transacetylase activity of rat liver when N-hydroxy-AABP was used as acetyl donor and N-hydroxy-4-aminobiphenyl (N-hydroxy-ABP) was the acetyl acceptor. This enzyme system from mammary tissue did not utilize the fluorene derivatives as either acetyl donor or acetyl acceptor, was much more labile than the liver, kidney, or intestinal mucosa systems, and had a pH optimum at 7.5, as compared to pH 6.8 for liver. The mammary tissue system was similar to the hepatic system in being inhibited by sulfhydryl reagents; it required a source of reduced pyridine nucleotides for maximum activity.     

The binding of metabolites formed from aminostilbene derivatives to nucleic acids in the liver of rats.

Carcinogenic trans-4-dimethylaminostilbene (trans-DAS) and trans-4-acetylaminostilbene (trans-AAS) as well as inactive cis-DAS and DABB were highly and specifically labeled with tritium and administered orally to female Wistar rats. Covalent binding to liver rRNA and DNA was measured and found to be higher for the carcinogenic compounds. Digests from these nucleic acids were chromatographed on Sephadex LH-20 and 16 different nucleoside adducts were characterised by their retention volumes. Labeled trans-DAS was administered in doses ranging from 0.025--250 mumol/kg. Binding to nucleic acids was directly proportional to the dose at low doses (0.025--2.5 mumol/kg) and less than proportional at higher doses (25--250 mumol/kg). The pattern of nucleoside adducts remained practically constant over the wide range of doses. A pharmacokinetically determined threshold of metabolic activation thus could not be demonstrated for this compound. A modified procedure is described to simultaneously isolate pure liver rRNA and DNA from nonfasted rats in high yields.     

Comparison of rat and guinea pig as sources of the S9 fraction in the salmonella/mammalian microsome mutagenicity test

A highly significant enhancement of mutagenicity occurs with 11 polycyclic aromatic hydrocarbons when 3-methylcholanthrene-induced guinea pig liver S9 is substituted for Aroclor-induced rat liver S9 in the Ames test. The use of MC-induced guinea pig liver S9 is particularly valuable for detecting the weak mutagenicity of benz[c]acridine, which is barely positive in a standard Ames assay. However, anthracene and phenanthrene, which are generally considered not to be carcinogens, remain non-mutagenic for strain TA100. This enhancement of mutagenicity does not correlate with arylhydrocarbon hydroxylase activities of the various liver preparations and does not apply to certain other non-PAH mutagens, including β-naphthylamine, aflatoxin B₁ and 4-dimethylaminoazobenzene.     

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of ¹²⁵I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of ¹²⁵I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin.Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10^?11 mol/l to 10^?6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10⁷ l/mol with a total receptor concentration of 3.4 · 10⁶ sites per cell.The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10^?9 mol/l), both, under conditions of low and high fractional saturation of receptors.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Studies on the mobilization of iron from ferritin by isolated rat liver mitochondria

Rat liver mitochondria and rat liver mitoplasts mobilize iron from ferritin by a mechanism which depends on a respiratory substrate (preferentially succinate), a small molecular weight electron mediator (FMN, phenazine methosulphate or methylene blue) and (near) anaerobic conditions.The release process under optimized conditions (approx. 50 μmol/l FMN, 1 mmol/l succinate, 0.35 mmol/l Fe(III) (as ferritin iron), 37°C and pH 7.40) amounts to 0.9–1.2 nmol iron/mg protein per min.The results suggest that ferritin might function as an intermediate in the cytosolic transport of iron to the mitochondria.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM⁻¹ · cm⁻¹) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min⁻¹ · mg⁻¹ of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.

Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

A comparison of the DNA binding,cytotoxicity and repair synthesis induced in human fibroblasts by reactive derivatives of aromatic amide carcinogens

The cytotoxicity of three structurally-related direct-acting carcinogens, N-acetoxy-2-acetylaminofluorene, N-acetoxy-2-acetylaminophenanthrene and N-acetoxy-4-acetylaminobiphenyl, was compared in normal cells and in excision repair deficient xeroderma pigmentosum cells (XP12BE). All three proved significantly more cytotoxic to the XP cells than to the normal cells. At equicytoxic levels, substantially more residues were initially bound to the DNA of the normal cells than to the XP cells, suggesting that the former are able to remove a large percentage of the DNA bound residues before these can result in cell death. The ability of these cell strains to remove bound residues from DNA, to incorporate thymidine into parental strands of DNA during repair replication, and to recover from potentially lethal damage if held in the non-replicating, density-inhibited G_o state was compared as a function of dose and time. The XP12BE cells proved virtually incapable of excision repair of DNA damage induced by these carcinogens and of recovery. In contrast, normal cells recovered from the potentially lethal effects of these three compounds and did so at a rate comparable to their rate of removal of bound residues and of repair synthesis. In the excision-deficient XP12BE cells, DNA adducts induced by N-acetoxy-2-acetylaminophenanthrene proved 3- to 6-fold more cytotoxic than adducts induced by the other two carcinogens.