Antiestrogen binding sites in rat liver nuclei

Isolated, intact rat liver nuclei have high-affiity (K_d=10⁻⁹ M) binding sites that are highly specific for nonsteroidal antiestrogens, especially for compounds of the triphenylethylene series. Nuclear [³H]tamoxifen binding capacity is thermolabile, being most stable at 4°C and rapidly lost at 37°C. More [³H]tamoxifen, however, is specifically bound at incubation temperatures of 25°C and 37°C than at 4°C although prewarming nuclei has no effect, suggesting exchange of [³H]tamoxifen for an unidentified endogenous ligand. Nuclear antiestrogen binding sites are destroyed by trypsin but not by deoxyribonuclease I or ribonuclease A. The nuclear antiestrogen binding protein is not solubilized by 0.6 M potassium chloride, 2 M sodium chloride, 0.6 M sodium thiocyanate, 3 M urea, 20 mM pyridoxal phosphate, 1% (w/v) digitonin or 2% (w/v) sodium cholate but is extractable by sonication, indicating that it is tightly bound within the nucleus. Rat liver nuclear matrix contains high-affinity (K_d=10⁻⁹ M) [³H]tamoxifen binding sites present in 5-fold higher concentrations (4.18 pmol/mg DNA) than in intact nuclei (0.78±0.10 (S.D.) pmol/mg DNA). Low-speed rat liver cytosol (20 000×g, 30 min) contains high-capacity (955±405 (S.D.) fmol/mg protein), low-affinity (K_d=10.9±4.5 (S.D.) nM) antiestrogen binding sites. In contrast, high-speed cytosol (100 000×g, 60 min) contains low-capacity (46±15 (S.D.) fmol/mg protein), high-affinity (K_d=0.61± 0.20 (S.D.) nM) binding sites. Low-affinity cytosolic sites constitute more than 90% of total liver binding sites, high-affinity cytosolic sites 0.3%–3.2%, and nuclear sites less than 0.5% of total sites.     

Complexation of lithium(I), sodium(I), silver(I) and thallium(I) by 4,7,13,16-tetraoxa-1,10-diazabicyclo[8.5.5]eicosane and 4, 7, 13-trioxa-1,10-diazabicyclo[8.5.5]eicosane in trimethyl phosphate. A potentiometric titration and Li and Na nuclear magnetic resonance study

Complexation of M⁺=Li⁺, Na⁺, Ag⁺ and TI⁺ by the cryptands 4, 7, 13, 18-tetraoxa-l, 10-diazabicyclo[8.5.5]eicosane (C211) and 4,7,13-trioxa-1,10-diazabicyclo[8.5.5]eicosane (C21C₅) to form the cryptates [M.C211]⁺ and [M.C21C₅]⁺ has been studied in trimethyl phosphate by potentiometric titration and ⁷Li and ²³Na NMR spectroscopy. For [M.C211]⁺ the logarithm of the apparent stability constants, log K (dm³ mol^-1)=6.98±0.05, 5.38±0.05, 9.82±0.02 and 3.95±0.02 for M⁺ =Li⁺, Na⁺, Ag⁺ and TI⁺, respectively; and for [M.C21C₅]⁺ log K (dm³ mol^-1)=2.40±0.10, 1.90±0.05, 6.04±0.02 and 2.42±0.10 for M⁺=Li⁺, Na⁺, Ag⁺ and Tl⁺, respectively. The decomplexation kinetic parameters for [Na.C211]⁺ are: k_d (298.2 K)=6.924±0.50 s^-l, ΔH_d≠=62.2±0.9 kJ mol^-1, and ΔS_d≠= -20.3±2.7 J K^-1 mol^-1; and those for [Li.C21C₅]⁺ are: k_d (298.2 K)=23.3±0.4 s^-1, ΔH_d≠ =61.2±1.1 kJ mol^-1, and ΔS_d≠= -13.6±3.6 J K^-1 mol^-1. Metal ion exchange on [Li.C211]⁺ is in the very slow extreme of the NMR timescale up to 390 K and k_d « 4 s^-1 at 298.2 K, while in contrast exchange on [Na.C21C₅]⁺ is in the fast extreme of the NMR timescale at 298.2 K (k_d≈ 10⁴ s^-1). These data are compared with those obtained in other solvents.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

Computer Modeling of the Solution Conformation of Cyclic Enkephalins

Summary The probable conformations of two cyclic enkephalin analogs, DNS-cyclo[d-Dab-Gly-Trp-Leu] (I) and DNS-cyclo[d-Dab-Gly-Trp-d-Leu] (II) (DNS=dansyl), were determined by combining the results of NOE, vicinal coupling constant and fluorescence energy transfer measurements with theoretical calculations. The common feature of the conformations for both peptides is the presence of a β-turn at residues 2 and 3.     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

A comparison of negatively and positively charged liposomes containing entrapped polynosinic · polycytidylic acid for interferon induction in mice

Intravenous injection of negatively charged liposomes containing entrapped poly(I) · poly(C) induced a vigorous interferon response in mice with serum titers of interferon reaching twenty times those observed with comparable dosages of free poly(I) · poly(C). The response did not persist over an extended time period as was observed earlier for enhanced interferon production stimulated by positively charged liposomes containing the inducer. Both negatively and positively charged liposomes containing [¹⁴C]poly(I) · poly(C) were taken up chiefly by the liver when given intravenously. Negatively charged particles were concentrated somewhat preferentially by the spleen (7–9% of the dose compared to 4–6%). Less radioactivity was found in liver and spleen when negatively charged particles were given intraperitoneally than was the case when positively charged particles were injected by this route. Free [¹⁴C]poly(I) · poly(C) was extensively metabolized to low molecular weight materials within four hours of injection, while encapsulation of the polymer provided protection against in vivo degradation. When both preferential localization and protection were considered, from three to five times as much high molecular weight [¹⁴C]poly(I) · poly(C) was recovered from liver at four hours after intravenous injection when the compound was given in encapsulated form compared to the free polymer. Similarly, for spleen, seven times and three times as much polymeric [¹⁴C]poly(I) · poly(C) was recovered following injection of negatively charged liposomes and positively charged liposomes respectively compared to free [¹⁴C]poly(I) · poly(C). At 48 h after an intravenous injection of positively charged liposomes, as much as four percent of the dose remained in high molecular weight form in the liver and one percent in the spleen. Following intraperitoneal injections, polymeric [¹⁴C]poly(I) · poly(C) recovered from the liver never exceeded 4.3% of the dose, showing that most of the radioactivity in the liver consisted of metabolites. These results suggest that elevated and prolonged production of interferon in animals treated with encapsulated inducer results from a combination of factors including preferential tissue location and protection of the inducer from hydrolytic cleavage.     

Studies on fungal polysaccharide XVII. A new glucuronan “protuberic acid” produced by a fungus Kobayashi Nipponica

A water-soluble glucuronan “protuberic acid”, [α]_d²² −83.6° and purified from Kobayashia Nipponica, and its physicochemical properties were investigated.The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1,4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [α]_d²² −44°, is also described.     

A structural transition in egg lecithin-cholesterol bilayers at 12 °C

The thermal coefficient of expansion of egg lecithin bilayer thickness, αd₁, was measured as a function of its cholesterol content up to mole ratio lecithin/cholesterol of 1:1, and over the temperature range 0–40 °C. At all cholesterol contents αd₁ changes abruptly at approximately 12 °C indicating a structural transition at this temperature. Above 12 °C, αd₁ decreases monotonically from −2·10⁻³ for pure egg lecithin to −1·10^–3 at mole ratio 1:1. Below 12 °C αd₁ is walways higher than above 12 °C and shows a sharp, anomalously high value of −6·10⁻³ at the mole ratio 2:1. The results have been interpreted as the movement of cholesterol into the bilayer or the formation of lecithin-cholesterol “complexes” at temperatures below 12 °C. Similar studies with phosphatidylinositol containing cholesterol showed no structural transition and lysolecithin containing cholesterol behaved differently giving two lamellar phases in equilibrium.     

Growth characteristics of flagellated cells of Phaeocystis pouchetii revealed by diel changes in cellular DNA content

The present study reports on effects of different light:dark periods, light intensities, N:P ratios and temperature on the specific growth rate of flagellated cells of Phaeocystis pouchetii in culture. The specific growth rate was estimated by diel changes in cellular DNA content. The cellular DNA content and cell cycle of flagellated cells of P. pouchetii are shown, and the importance of light:dark period in cell division is demonstrated. Diel patterns of the cellular DNA content showed that cell division was confined to the dark period. The cells dealt with more than one division per day by rapid divisions shortly after each other.The specific growth rates (μ_DNA) based on the DNA cell cycle model were in close agreement with specific growth rates (μ_Cell) determined from cell counts. The temperature affected the specific growth rates (multiple regression, p < 0.01) and were higher at 5 °C (μ ≤ 2.2 d⁻¹) than at 10 °C (μ ≤1.6 d⁻¹). Increasing the light:dark period from 12:12 h to 20:4 h affected the specific growth rate of P. pouchetii at the lower temperature tested (5 °C) (multiple regression, p < 0.01), resulting in higher specific growth rates than at 10 °C. At 10 °C, the effect of light:dark period was severely reduced. Neither light nor nutrients could compensate the reduction in specific growth rates caused by elevated temperature. The specific growth rates was not affected by the N:P ratios tested (multiple regression, p = 0.21). The experiments strongly suggest that the flagellated cells have a great growth potential and could play a dominating role in northern areas at increased day length.     

7-Amino-actinomycin D complexes with deoxynucleotides as models for the binding of the drug to DNA

Fluorescence, CD, absorption, and ¹H-nmr studies are reported for complexes of 7-amino-actinomycin D with deoxydinucleotides, deoxytetranucleotides, and poly(dG-dC)· poly(dG-dC). The optical spectra for the 7-amino-actinomycin D complex with pdG-dC, pdG-dC-dG-dC and pdC-dG-dC-dG are similar in shape to the 7-amino-actinomycin D complex with either DNA or poly(dG-dC). The changes in the ¹H chemical shifts of the 7-amino-actinomycin D and the pdG-dC resonances that accompany complex formation show that 7-amino-actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The magnitudes of the induced chemical shifts for the 7-amino-actinomycin D complex formation with pdG-dC are similar to, but slightly different from, the induced chemical shifts which are obtained when actinomycin D forms a minature intercalated complex with two pdG-dC molecules. The pdN-dG dinucleotides (N = C, A, or T) form stacked complexes with 7-amino-actinomycin D. The presence of the 7-amino-group results in a larger dimerization constant (in aqueous solution) for 7-amino-actinomycin D [K_D(6°C) = 4.4 × 10³M^?1], as compared to actinomycin D [K_D(6°C) = 1.7 × 10³M^?1]; the chemical shifts which accompany dimer formation indicate that the chromophores stack in an inverted manner. Intercalation of 7-amino-actinomycin D into minature double helices, as well as into calf thymus DNA, poly(dG-dC)·poly(dG-dC), and poly(dA-dC)·poly(dG-dT), results in an enhancement of the relative fluorescence intensity and a shift in both the absorbance and corrected emission spectra.     

Melanoma targeting with α-melanocyte stimulating hormone analogs labeled with fac-[99mTc(CO)3]+: effect of cyclization on tumor-seeking properties

Early detection of primary melanoma tumors is essential because there is no effective treatment for metastatic melanoma. Several linear and cyclic radiolabeled α-melanocyte stimulating hormone (α-MSH) analogs have been proposed to target the melanocortin type 1 receptor (MC1R) overexpressed in melanoma. The compact structure of a rhenium-cyclized α-MSH analog (Re-CCMSH) significantly enhanced its in vivo tumor uptake and retention. Melanotan II (MT-II), a cyclic lactam analog of α-MSH (Ac-Nle-cyclo[Asp-His-dPhe-Arg-Trp-Lys]-NH₂]), is a very potent and stable agonist peptide largely used in the characterization of melanocortin receptors. Taking advantage of the superior biological features associated with the MT-II cyclic peptide, we assessed the effect of lactam-based cyclization on the tumor-seeking properties of α-MSH analogs by comparing the pharmacokinetics profile of the ^99mTc-labeled cyclic peptide βAla-Nle-cyclo[Asp-His-d-Phe-Arg-Trp-Lys]-NH₂ with that of the linear analog βAla-Nle-Asp-His-dPhe-Arg-Trp-Lys-NH₂ in melanoma-bearing mice. We have synthesized and coupled the linear and cyclic peptides to a bifunctional chelator containing a pyrazolyl-diamine backbone (pz) through the amino group of βAla, and the resulting pz–peptide conjugates were reacted with the fac-[^99mTc(CO)₃]⁺ moiety. The ^99mTc(CO)₃-labeled conjugates were obtained in high yield, high specific activity, and high radiochemical purity. The cyclic ^99mTc(CO)₃-labeled conjugate presents a remarkable internalization (87.1% of receptor-bound tracer and 50.5% of total applied activity, after 6 h at 37 °C) and cellular retention (only 24.7% released from the cells after 5 h) in murine melanoma B16F1 cells. A significant tumor uptake and retention was obtained in melanoma-bearing C57BL6 mice for the cyclic radioconjugate [9.26 ± 0.83 and 11.31 ± 1.83% ID/g at 1 and 4 h after injection, respectively]. The linear ^99mTc(CO)₃-pz–peptide presented lower values for both cellular internalization and tumor uptake. Receptor blocking studies with the potent (Nle⁴,dPhe⁷)-αMSH agonist demonstrated the specificity of the radioconjugates to MC1R (74.8 and 44.5% reduction of tumor uptake at 4 h after injection for cyclic and linear radioconjugates, respectively).     

Determination of extracellular and intracellular enrichments of [1-C]-α-ketoisovalerate using enzymatically converted [1-C]-valine standardization curves and gas chromatography–mass spectrometry

We report a validated method for the determination of extra- and intracellular [1-¹³C]-α-ketoisovalerate ([1-¹³C]-KIV) enrichments by gas chromatography–mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-¹³C]-valine enriched standards of known composition. Slopes of [1-¹³C]-valine standardization curves (mean±SD: 0.99±0.02, n=5) and [1-¹³C]-KIV standardization curves (mean±SD: 0.98±0.01, n=7) were not significantly different. The method was applied for the determination of [1-¹³C]-KIV enrichments in plasma and tissues during [1-¹³C]-valine infusion in a piglet. [1-¹³C]-KIV enrichment could be determined±0.1 MPE (C.V. 1%), and extracellular [1-¹³C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-¹³C]-KIV enrichment.     

Dopamine-stimulated cyclic AMP and binding of [H]dopamine in acini from dog pancreas

Dispersed acini from dog pancreas were used to examine the ability of dopamine to increase cyclic AMP cellular content and the binding of [³H]dopamine. Cyclic AMP accumulation caused by dopamine was detected at 1·10⁻⁸ M and was half-maximal at 7.9±3.4·10⁻⁷M. The increase at 1·10⁻⁵ M, (7.5-fold) was equal to the half-maximal increase caused by secretin at 1·10⁻⁹ M. Haloperidol, a dopaminergic receptor antagonist inhibited cyclic AMP accumulation caused by dopamine. The IC₅₀ value for haloperidol, calculated from the inhibition of cyclic AMP increase caused by 1·10⁻⁵ M dopamine was 2.3±0.9·10⁻⁶M. Haloperidol did not alter basal or secretin-stimulated cyclic AMP content. [³H]Dopamine binding was studied on the same batch of cells as cyclic AMP accumulation. At 37°C, it was rapid, reversible, saturable and stereospecific. The K_d value for high affinity binding sites was 0.43±0.1·10⁻⁷M and 4.7±1.6·10⁻⁷M for low affinity binding sites. The concentration of drugs necessary to inhibit specific binding of dopamine by 50% was 1.2±0.4·10^/t-7M noradrenaline, 2·10^/t-7 M epinine, 4.1±1.8·10^/t-6M fluphenazine, 8.0±1.6·10^/t-6M haloperidol, 4.2±1.2·10⁻⁶Mcis-flupenthixol, 2.7±0.4·10⁻⁵Mtrans-flupenthixol, >1·10⁻⁵M apomorphine, sulpiride, naloxone and isoproterenol.     

Inactivation of de novo DNA methyltransferase activity by high concentrations of double-stranded DNA

The activity of eukaryotic DNA methyltransferase diminishes with time when the enzyme is incubated with high concentrations (200–300 μg/ml) of unmethylated double-stranded Micrococcus luteus DNA. Under similar conditions, single-stranded DNA induces only a limited decrease of enzyme activity. The inactivation process is apparently due to a slowly progressive interaction of the enzyme with double-stranded DNA that is independent of the presence of S-adenosyl-l-methionine. The inhibited enzyme cannot be reactivated either by high salt dissociation of the DNA-enzyme complex or by extensive digestion of the DNA. Among synthetic polydeoxyribonucleotides both poly(dG-dC) · poly(dG-dC) and poly(dA-dT) · poly(dA-dT), but not poly(dI-dC) · poly(dI-dC), cause inactivation of DNA methyltransferase. This inactivation process may be of interest in regulating the ‘de novo’ activity of the enzyme.     

Rate of DNA synthesis as a function of temperature in cultured hamster fibroblasts (V-79) and HeLa-S3 cells

The rates of intracellular DNA synthesis at various temperatures between 39 ° and 31 °C were determined in hamster fibroblasts and HeLa cells by measuring average amounts of ³H-thymidine incorporated per cell in S phase per unit of time. The energy of activation and Q₁₀ for intracellular DNA synthesis were calculated from the slopes of the relative rates of DNA synthesis in HeLa cells and hamster fibroblasts vs. time, plotted on Arrhenius coordinates. In both cell types the incorporation of thymidine into DNA is characterized by an energy of activation of 21 000 calories/mole and a Q₁₀ of 2.94. The absolute rates of DNA synthesis were determined in hamster cells at various temperatures, with values ranging from 1.44 to 0.60 × 10⁻¹⁴ g DNA/ min/cell at 39 ° to 31 °C, respectively. The length of the S phase of the hamster cell was calculated over a 39 ° to 31 °C range, and found to be 5.0 to 11.9 h, respectively. It is concluded that the S phase length is partly determined by the rate of temperature-dependent DNA synthesis.     

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-¹⁴C]d-(–)-hydroxybutyric acid, [1-¹⁴C]d-lactic acid, [1-¹⁴C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(–)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-¹⁴C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-¹⁴C]lactyl-CoA was used as a substrate at a rate that was only less than 10^–4 of the rate than with [3-¹⁴C]d-(–)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-¹⁴C]d-lactyl-CoA as substrate at a relatively high rate. Correspondence to: A. Steinbüchel     

Light or high (30°C) temperature effects on l-[U-C]leucine incorporation and protein patterns in embryos and endosperm during the early phase of germination of the negatively photoblastic and thermosensitive seeds of Phacelia tanacetifolia

The activities of l-[U-¹⁴C]leucine uptake and incorporation into proteins of embryos and endosperm of seeds of Phacelia tanacetifolia Benth. cv Bleu-Clair were analysed during the first 24 h of incubation under conditions optimal for germination (16°C in darkness) and in two inhibitory conditions: 16°C in the light and 30°C in darkness. Blocking germination induced by light or 30°C was accompanied by the inhibition of l-[U-¹⁴C]leucine uptake and incorporation in embryos. In the endosperm, the activation of l-[U-¹⁴C]leucine uptake was of the same magnitude for the non-inhibited and the light-inhibited seeds and much higher for the 30°C-inhibited seeds; the activation of l-[U-¹⁴C]leucine incorporation was quantitatively similar in all three conditions, with the patterns of newly synthesised proteins qualitatively different in the endosperm from light- or 30°C-inhibited seeds. The results showed that germination of P. tanacetifolia seeds is controlled by light or super-optimal temperature through the inhibition of the activation of transport and protein synthetic activities in embryo without effect on the endosperm. We suggest, on the basis of the translational activity, the possibility that in the inhibitory conditions the blockade of the embryo to operate as a sink affects the transition of the endosperm to operation as a source.     

Reaction of apurinic/apyrimidinic sites with [C]methoxyamine: A method for the quantitative assay of AP sites in DNA

A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of [¹⁴C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37°C, pH 7.2) untreated DNA is almost unreactive and the [¹⁴C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.     

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.     

Toxicity and mutagenicity of X-rays and [I]dUrd or [H]TdR incorporated in the DNA of human lymphoblast cells

We measured the toxicity and mutagenicity induced in human diploid lymphoblasts by various radiation doses of X-rays and two internal emitters. [¹²⁵I]iododeoxyuridine ([¹²⁵I]dUrd) and [³H]thymidine ([³H]TdR), incorporated into cellular DNA. [¹²⁵I]dUrd was more effective than [³H]TdR at killing cells and producing mutations to 6-thioguanine resistance (6TG^R). No ouabain-resistant mutants were induced by any of these agents. Expressing dose as total disintegrations per cell (dpc), the D₀ for cell killing for [¹²⁵I]dUrd was 28 dpc and for [³H]TdR was 385 dpc. The D₀ for X-rays was 48 rad at 37°C. The slopes of the mutation curves were approximately 75 × 10⁻⁸ 6TG^R mutants per cell per disintegration for [¹²⁵I]dUrd and 2 × 10⁻⁸ for [³H]TdR. X-Rays induced 8 × 10⁻⁸ 6TG^R mutants per cell per rad. Normalizing for survival, [¹²⁵I]dUrd remained much more mutagenic at low doses (high survival levels) than the other two agents. Treatment of the cells at either 37°C or while frozen at −70°C yielded no difference in cytotoxicity or mutation for [¹²⁵I]dUrd or [³H]TdR, whereas X-rays were 6 times less effective in killing cells at −70°C.Assuming that incorporation was random throughout the genome, the mutagenic efficiencies of the radionuclides could be calculated by dividing the mutation rate by the level of incorporation. If the effective target size of the 6TG^R locus is 1000–3000 base pairs, then the mutagenic efficiency of [¹²⁵I]dUrd is 1.0–3.0 and of [³H]TdR is 0.02–0.06 total genomic mutations per cell per disintegration. ¹²⁵I disintegrations are known to produce localized DNA double-strand breaks. If these breaks are potentially lethal lesions, they must be repaired, since the mean lethal dose (D₀) was 28 dpc. The observations that a single dpc has a high probability of producing a mutation (mutagenic efficiency 1.0–3.0) would suggest, however, that this repair is extremely error-prone. If the breaks need not be repaired to permit survival, then lethal lesions are a subset of or are completely different from mutagenic lesions.