Metabolism of [2-14C]thymine and [2-14C]thymidine in germinating black gram (Phaseolus mungo) seeds

The metabolism of [2-14C]thymine and [2-14C]thymidine in the cotyledons and embryonic axes of black gram (Phaseolus mungo) seedlings was investigated. Both [2-14C]thymine and [2-14C]thymidine degraded extensively into [14C]CO2. The rate of release of [14C]CO2 from [2-14C]thymine was much greater than that from [2-14C]thymidine. Radioactivity from both precursors was also observed beta-ureidoisobutyric acid. This indicated that thymine was degraded by the reductive pathway of pyrimidine degradation. Small amounts of [2-14C]thymine and [2-14C]thymidine were salvaged for deoxyribonucleotide and DNA synthesis. The highest incorporation of [2-14C]thymine and [2-14C]thymidine into the DNA fraction was observed in 24 hour-old cotyledons where net DNA synthesis was not observed. These precursors seem to be utilised for DNA synthesis of organelles of the cotyledonary cells, probably mitochondria. In embronic axes, [2-14C]thymine is more effectively salvaged for DNA synthesis than [2-14C]thymine. The incorporation rate increased during the early phase of germination and attained its maximum at 48 h after which it decreased. No thymidine kinase activity was detected in either cotyledons or in the embryonic axes. Thymidine salvage seems to be catalysed by nucleoside phosphotransferase which is present both in the cotyledons and in the embryonic axes. This suggests that, in contrast to other pyrimidine and purine bases and nucleosides, no specific salvage system for thymine and thymidine is present in black gram seedlings.     

5-Hydroxymethyl-2'-deoxyuridine. Cytotoxicity and DNA incorporation studied by using a novel [2-14C]-derivative with normal and leukemic human hematopoietic cells

5-Hydroxymethyl-2'-deoxyuridine is a biologically active thymidine analogue. This investigation was aimed at characterizing the cytotoxicity of 5-hydroxymethyl-2'-deoxyuridine and its incorporation into DNA. Fifty percent inhibition of cellular proliferation, assessed by incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) incorporation of [U-14C]-L-leucine in vitro, was caused by 1.7-5.8 X 10(-5) M 5-hydroxymethyl-2'-deoxyuridine in seven human leukemia cell lines. Higher concentrations of 5-hydroxymethyl-2'-deoxyuridine, i.e. 6-8 X 10(-5) M, were required for a comparable inhibition in human PHA-stimulated peripheral blood lymphocytes. A new synthesis procedure for [2-14C]5-hydroxymethyl-2'-deoxyuridine was developed. The net incorporation of [2-14C]5-hydroxymethyl-2'-deoxyuridine into DNA of hematopoietic cells was low. The possibility of a repair mechanism for 5-hydroxymethyluracil bound to DNA is discussed.     

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidin Earlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3) . To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12-[³H]-farnesol as an internal standard: 2-[¹⁴C]-, 7-[¹⁴C]-, 7′, 8-[¹⁴C]-, 7′, 8-[³H]-geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U-[¹⁴C]-leucine, U-[¹⁴C]-isoleucine and 1-[¹⁴C]-glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1-[¹⁴C]- and 2-[¹⁴C]-glycine do not serve as precursors for cantharidin (1) .     

On the biosynthesis of dendrolasin, a body constituent of the ant Lasius fuliginosus Latr

By feeding the ant Lasius fuliginosus LATR . with [¹⁴C]-1-acetate, [¹⁴C]-2-acetate, [¹⁴C]-2-mevalonate, [¹⁴C]-2-mevalonate, [¹⁴C]-1-glucose and [¹⁴C]-U-glucose, incorporation ratios of 10^?4 – 0,15% were obtained in the sesquiterpenoid dendrolasin. It was shown by analysis of the labelling pattern in dendrolasin that the insertions were spread over the whole molecule in exactly the manner that would be expected from terpene biosynthesis.     

5-Ethyl-2'-deoxyuridine. Cytotoxicity and DNA incorporation demonstrated with human leukemic cells and PHA-stimulated lymphocytes in vitro

5-Ethyl-2'-deoxyuridine (5EtdUrd) is a biologically active thymidine analogue. The cytotoxicity of 5EtdUrd was investigated with seven established human leukemia cell lines as well as with human peripheral blood PHA-stimulated lymphocytes. All types of leukemia cells were susceptible to the toxicity of 5EtdUrd as assayed with a [U-14C]-L-leucine incorporation system developed for this study. A 50% inhibition of leucine incorporation in 3-day cultures was induced by 1.3-3.8 microM 5EtdUrd with leukemic cells, but the concentration required to induce similar inhibition with PHA-stimulated lymphocytes was approximately was approximately 100-fold. The toxicity of 5EtdUrd seemed to require active DNA synthesis, since the inhibition of leucine incorporation became obvious only after the first 24 hours of culture. The DNA incorporation studies were based on a new isotopically labeled 5EtdUrd derivative, [2-14C]5EtdUrd, synthesized for this study in our laboratory. It was demonstrated for the first time that most of the radioactivity derived from [2-14C]5EtdUrd in DNA was in 5-ethyluracil. 5EtdUrd has a powerful antileukemic potency in vitro. Its effects against human leukemia in vivo remain to be tested.     

Structural changes in myosin during contraction and the state of ATP in the intact frog muscle.

The reactivity of myosin to [14C]-labeled N-ethylmaleimide ([14C]NEM) or to tritium was determined in functionally different frog muscles. The incorporation of [14C]NEM into myosin decreased during isotonic or isometric contractions, as compared to resting muscle. The cysteine residues which were protected during contraction were not involved in the ATPase activity or the actin-binding ability of myosin. Peptide mapping revealed that several residues were protected simultaneously. The incorporation of tritium into the peptide N-H groups of myosin was also decreased during muscle activity. These data support the idea that activation and subsequent contraction of muscle are correlated with structural changes in the myosin molecule. The reactivity of myosin to [14C]NEM was increased when the muscle was stretched to 140% rest length and treated with iodoacetate to deplete ATP. Based on in vitro experiments and on literature data, it is suggested that in the resting muscle myosin contains bound MgATP which decreases the rate of incorporation of [14C]NEM into myosin and that upon the irreversible loss of ATP the rate increases. 31P nuclear magnetic resonance signals from a number of phosphates were detected in the intact frog muscle. The data indicated that the minimum concentration of ATP in the muscle is 3 mM, a value which agrees with that of chemical determination. The characteristic chemical shifts, coupling constants, and line widths of ATP in the muscle were considerably altered from that of either free ATP in aqueous solutions or ATP in perchloric acid extracts of muscle.     

Biological activities of phthalocyanines. XII: Synthesis tumor uptake and biodistribution of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine in C3H mice

The biodistribution and metabolism of 14C-labeled disulfonated and trisulfonated gallium phthalocyanine (Ga-PcS) was studied in radiation-induced fibrosarcoma tumor-bearing C3H mice. The [14C]Ga-PcS compounds were prepared via the condensation of [14C]phthalic acid and sulfophthalic acid in the presence of gallium chloride and characterized by their spectroscopic and chromatographic properties. The tissue concentrations of the dyes was measured by scintillation counting of the 14C and by extraction and fluorescence measurements. Elevated dye levels were found in the liver, lungs, kidneys and spleen as well as in the tumor. Lower sulfonation of Ga-PcS favored liver and spleen uptake whereas higher dye sulfonation resulted in greater kidney uptake. Both dyes showed high tumor uptake with peak concentrations exceeding those of most tissues except for the liver in the case of Ga-PcS2. The highest tumor uptake was observed with Ga-PcS3. Both dyes were slowly excreted from the body. The liver-feces pathway was favored in the case of Ga-PcS2 with high activities persisting in the liver, even after 21 days. The Ga-PcS3 was preferentially excreted via the kidney-urine pathway. High performance liquid chromatography analysis of the liver and tumor extracts of [14C]Ga-PcS3-treated animals did not reveal desulfonation of the dye. However, urine analysis showed the presence of radioactive metabolites lacking the characteristic phthalocyanine absorption.     

Synthetic approaches to phomactins: Late stage stereochemical and reactivity issues

Oxidation of a 15-methylenebicyclo[9.3.1]pentadeca-3,7-dien-14-ol with a 10-phenylsulfonyl substituent provided a 14-oxobicyclo[9.3.1]pentadeca-1(15),3,7-triene-15-carbaldehyde that on reduction with DIBAL-H was converted into a 15-hydroxymethylbicyclo[9.3.1]pentadeca-1(15),3,7-triene-14-ol with the relative configuration at C14 required for incorporation into a synthesis of phomactin A. The oxidation and reduction of an analogous 3,4-epoxide that lacked the 10-phenylsulfonyl group gave a diol with the opposite relative configuration at C14. However, the TPAP oxidation and DIBAL-H reduction of a 14-hydroxy-15-methylene-3,4-epoxide that still had the 10-phenylsulfonyl group gave a diol with the required configuration at C14 although the expected spontaneous participation of the 14-hydroxyl group in an intramolecular epoxide ring-opening was only observed under Lewis acidic conditions.     

Einbauversuche mit (3H und 14C)-doppelmarkiertem Farnesol in Cantharidin. 5. Mitteilung zur Biosynthese des Cantharidins

Incorporation experiments with (³H and ¹⁴C) doubly labelled farnesols into cantharidin After injection of 11′, 12-[³H]-7-[¹⁴C]-farnesol or 11′, 12-[³H]-5,6-[¹⁴C]-farnesol, the ³H-label is located specifically in the C(9)-methyl-group of cantharidin, whereas the ¹⁴C-labelling pattern follows an incorporation via acetic acid (Scheme 4). C-Atoms 5, 6 and 7 from the middle part of the farnesol molecule are utilized for cantharidin biosynthesis to an extent that is about 2.1–11% of the incorporation rate of the methyl groups C(11′) and C(12), depending on the position of the ¹⁴C-label in farnesol. These results confirm our earlier hypothesis [1] that the C₁₀-molecule cantharidin is biosynthesized from the C₁₅-precursor farnesol which is cleaved between C(1)–C(2), C(4)–C(5), and C(7)–C(8). The synthesis of 7-[¹⁴C]-farnesol and of 5,6-[¹⁴C]-farnesol is described.     

[C14MIM]Br离子液体对聚丙烯超临界CO2发泡性能的影响

本文对PP/[C₁₄MIM]Br体系的熔融过程和对CO₂的吸收, 以及[C₁₄MIM]Br在PP基体中的分散状态进行了研究, 并初步考察了[C₁₄MIM]Br对PP发泡性能的影响.     

PHOTOSENSITIZATION BY PHOTOFRIN II DELIVERED TO WI26VA4 SV40-TRANSFORMED HUMAN FIBROBLASTS BY LOW DENSITY LIPOPROTEINS: INHIBITION OF LIPID SYNTHESIS AND FATTY ACID UPTAKE

Irradiation with 365 nm light of Wi26VA4 SV40-transformed human fibroblasts cultured for 24 h in the presence of low density lipoproteins loaded with the anticancer porphyrin mixture Photofrin II resulted in a near complete inhibition of [14C]oleic acid incorporation into triacylglycerols, cholesteryl esters and phospholipids. More than 80% reduction of the fatty acid incorporation in all lipid classes was observed following an irradiation dose of 1 J/cm2. The activities of the respective acyltransferases, measured in vitro on cell homogenates, were also markedly diminished, but to a lesser extent than lipid synthesis from oleic acid. Moreover, oleic acid uptake by cells was strongly and rapidly reduced. It is suggested that the rapid inhibition of membrane phospholipid synthesis upon cell photosensitization, due to both a direct inactivation of acyltransferases and to a reduction of fatty acid utilization, could play an important role in the photocytotoxic effect of Photofrin II.     

Determination of 15NH3 by gas chromatography-mass spectrometry. Application to the measurement of putrescine oxidation by human plasma

A convenient and sensitive method for the determination of 15NH3 has been developed. Ammonia was purified from sample solutions by a modified microdiffusion method, derivatized with pentafluorobenzoyl chloride to pentafluorobenzamide (PFBA) and determined by gas chromatography-mass spectrometry using a multiple ion detector. PFBA was eluted from the gas chromatographic column within 2 min and resulted in a simple mass fragmentation pattern. The 15N/14N ratio was accurately determined with picomole amounts of PFBA by measuring the molecular ions of PFBA and [15N]PFBA. The method was applied to the assay of putrescine oxidation by human plasma. 15NH3 was produced by incubating 15N-labelled putrescine with plasma. The 15NH3 production was time dependent and strongly inhibited by the addition of aminoguanidine, an inhibitor of diamine oxidase. Exceedingly high 15NH3 production from [15N]putrescine was observed in the plasma from pregnant women. In contrast, only trace amounts of 15NH3 were formed in the plasma from normal men and non-pregnant women. The method seems to be applicable to various biological systems that produce ammonia as a metabolic product.     

Mo16O53F2]12-: a new polyoxofluoromolybdate anion

Single crystals of a new beta-octamolybdate salt containing protonated 1,4-diazabicyclo[2.2.2]octane cations were prepared under mild hydrothermal conditions. This compound, [C6H13N2]2[C6H14N2][Mo8O26], was then used as a starting material in the synthesis of [C6H13N2]6[Mo16O53F2].4H2O, which contains previously unreported [Mo16O53F2]12- anions. The structure-directing properties of gamma-[Mo8O26]4-, a likely intermediate in this pH-dependent transformation, are responsible for the site selection of the fluoride incorporation. [Mo16O53F2]12-, the largest reported polyoxofluoromolybdate cluster, expands upon the limited number of such anions in the literature. The structures of both compounds were determined using single-crystal X-ray diffraction.     

Biosynthesis of verrucarins and roridins. 3. Incorporation of 3R)-(5-14C)-and at C(2)stereospecific tritiated mevalonate into verrucarol]

Separate experiments, involving incorporation of either (3R)-[5-¹⁴C]-mevalonate or each enantiomer of [2-³H]-mevalonate into verrucartin A and roridin A, indicate that hydroxylation at C(4) of the tricothecane skeleton, to yield verrucarol (4), proceeds with an overall retention of configuration; they confirm that C(8) and not C(10) is derived specifically from C(2) or mevalonate.     

Syntheses of [prolyl-U-14C]alacepril and its related compounds

In order to study the metabolic fate of alacepril, an anti-hypertensive agent, the 14C-labeled compound of alacepril and its related compounds were synthesized. [Prolyl-U-14C]alacepril was synthesized in over-all yield of 32.7-38.0% by the mixed anhydride condensation of L-phenylalanine with [prolyl-U-14C]DU-1163, which had been prepared from L-[U-14C]proline and N-(S-3-acetylthio-2-methylpropanoyloxy)succinimide. [Prolyl-U-14C]captopril and [prolyl-U-14C]DU-1227 were prepared in high yields by hydrolysis of [prolyl-U-14C]DU-1163 and [prolyl-U-14C]alacepril, respectively. [Prolyl-U-14C]captopril-cysteine was synthesized by condensation of [prolyl-U-14C]captopril with cystine S-monoxide in 55.0% yield.     

Biological evaluation of radiolabeled D-methionine as a parent compound in potential nuclear imaging

Based on the high radioactivity uptake of some 14C-labeled D-amino acids in tumors and pancreas of mice, this investigation was undertaken to ascertain possible usefulness of radioactive D-methionine as a nuclear imaging agent. Radioactivity derived from D-[3,4-14C]-methionine in Ehrlich solid tumor was approximately three times higher than that from the L-isomer, but was the same as for mouse pancreas. Excretion rate of the radioactivity of D-[14C]-methionine into urine was approximately two times faster than that of the L-isomer. As compared with two potential imaging agents, 1-aminocyclopentane-1-carboxylic acid and alpha-aminoisobutyric acid, D-methionine showed almost the same radioactivity biodistribution in tumor and pancreas. These results suggest potential for D-methionine as a mother compound for use in tumor and pancreas imaging.     

An improved method for 14C-labelling of farnesylacetic acid and its geranyl ester

Farnesylacetic acid was efficiently labelled with 14C at the 5-position and gefarnate, a potent ulcer inhibitor, was prepared from it in radioactive form for use in metabolic studies. Condensation of [carbonyl-14C]acetyl chloride (5) with t-butyl 2-ethoxymagnesiomalonate (6) followed by acid-catalyzed deprotection and decarboxylation gave ethyl 3-oxo[3-14C]butanoate (8). Alkylation of the keto ester (8) with geranyl bromide (9) afforded the unsaturated keto ester (10), which was hydrolyzed and decarboxylated to give geranyl[2-14C]acetone (11). Grignard reaction of 11 with cyclopropylmagnesium bromide followed by treatment with hydrobromic acid yielded [4-14C]homofarnesyl bromide (13). Cyanation of 13 with potassium cyanide and subsequent hydrolysis gave [5-14C]farnesylacetic acid (1) in 6.1% yield from barium [14C]carbonate (3). Chlorination of 1 followed by esterification with geraniol afforded [5-14C]gefarnate (2) in 88% yield.     

Synthesis of (S)-N-[methyl-11C]nicotine and its regional distribution in the mouse brain: a potential tracer for visualization of brain nicotinic receptors by positron emission tomography.

A nicotine agonist, 11C-labeled (S)-nicotine, was synthesized by N-methylation of (S)-nornicotine with [11C]-methyl iodide in dimethylformamide-dimethylsulfoxide in order to study nicotinic receptors in the human brain by positron emission tomography. The radiochemical yield of this N-methylation reaction was more than 90% within 5 min. After purification by high performance liquid chromatography the radiochemical purity of the product was more than 99% and the specific radioactivity was 7.4-11.1 GBq/mumol. The regional distribution of (S)-[11C]nicotine in the mouse brain after intravenous injection was compared with that of (R)-[11C]nicotine. After injection of (S)-[11C]nicotine, the regional uptake of radioactivity was in the following order: cortex greater than thalamu approximately hippocampus greater than striatum greater than hypothalamus greater than cerebellum. Moreover, (S)-[11C]nicotine was displaced from the brain by unlabeled (S)-nicotine, but unlabeled (R)-nicotine caused no change in uptake. In contrast, (R)-[11C]nicotine showed a lower brain uptake and lesser regional differences in radioactivity.     

DNA EXCISION REPAIR IN LYMPHOCYTES FOLLOWING PHOTOAFFINITY LABELING WITH ETHIDIUM MONOAZIDE

Irradiation of ethidium monoazide by fluorescent light promotes a chemical decomposition of the azide into a highly reactive nitrene intermediate. Covalent bonding of this electrophile to the DNA in the cell provokes repair of damage which can be monitored by incorporation of [³H]-thymidine. Human lymphocytes were labeled with [¹⁴C]-ethidium azide and then allowed to undergo DNA repair. Repair incorporation of [³H]-thymidine showed saturation at 5 µM ethidium azide, but excision of the labeled drug failed to saturate at 20 µM, suggesting that excision and resynthesis are two separate events. Cells were also labeled with the photosensitive drug and/or exposed to UV radiation, and then allowed to undergo a period of DNA repair. The tritium incorporation for the combined insults was less than the sum of the two insults. Quinacrine, progesterone and chloroquine inhibited repair incorporation of [³H]-thymidine, but had no effect on the excision of the drug from the DNA. After damage by ethidium azide, chromatin was isolated from lymphocytes which had been allowed to repair label with [³H]-thymidine. Partial digestion of the chromatin with micrococcal nuclease released 80% of the tritium when approximately 40% of the DNA had been hydrolyzed by the enzyme.     

Zur Biosynthese der Rubratoxine

In order to check the hypothesis that rubratoxin B ( 2 ), a C₂₆-metabolite, is formed biogenetically by head-to-tail coupling of two identical C₁₃-precursors derived from decanoic acid and oxaloacetic acid, two labelled forms of the postulated C₁₃-intermediate 2-((E)-1'-octenyl)-3-[¹⁴C]methyl- and 2-((E)-1'-octenyl)-3-[¹³C]-methylmaleic anhydride ( 10 ), were synthesized. The labelled compounds 10 as well as a number of other ¹⁴C]- and [¹³C]-labelled potential precursors were administered to growing cultures of Penicillium rubrum STOLL . Significant incorporation rates of acetate (as intact units) and malonate were observed. Propionate was incorporated after decarboxylation. Succinate exhibited the highest rate of incorporation. The results are in agreement with the assumption that the C₁₀-chain is formed by the fatty acid pathway and the C₃-unit via the tricarboxylic acid cycle. After administration of 10 randomization of the label was observed. Thus the question whether compound 10 is a biogenetic intermediate remains unanswered.