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Mouse kidney and liver were found to increase their levels of radioactivity above that of serum from 2 to 60 min after administration of [6-14C]orotic acid. In spleen, thymus and brain, the radioactivity level reached a maximum soon after the injection and then decreased, as did that in serum. Sixty minutes after the injection, 44% of the administered isotope dose was found in the kidneys, 22% in the liver and 0.75% in the spleen. The 14C activity in liver UTP increased rapidly and then remained constant for 60 min. The ratio between the activities in uridine phosphates and UDP-sugars was 3:4 from 10- 60 min after injection. In the liver and kidneys, the RNA 14C activities at 60 min after injection were 15% of the activity in their acid-soluble fractions. Intraperitoneal administration was found to be preferable to intravenous administration for studies on nucleotides and RNA in mouse liver, due to the delayed incorporation of the [14C]orotic acid activity into the nucleotide pool.

The nucleoti-e contents of isolated rat Kupffer cells were found to be smaller than those of hepatocytes. The rate of UDPGal formation from D-galactose was much lower in Kupffer cells than in hepatocytes. The viability of the former was checked by measuring the leakage of enzymes and the formation of UTP from uridine. Addition of GalN to isolated Kupffer cells did not decrease their UTP and UDPG contents as much as those of hepatocytes. The same results were obtained when cells were isolated from GalN-pretreated animals. The incorporation of labeled amino acids into protein after GalN addition was much less reduced in Kupffer cells than in hepatocytes. The data suggest that Kupffer cells do not contribute to GalN-induced liver injury as a result of uridylate trapping.

Inhibition of IMP dehydrogenase in AS-30D hepatoma cells in suspension culture resulted in a pronounced and selective reduction of guanine nucleotide pools. Total acid-soluble guanine nucleotides decreased to 40% and the content of GTP and GDP dropped to about 20% of control within 4 h when mycophenolate or ribavirin were used as the inhibitors. Induction of GTP deficiency was associated with a 50% rise in UTP and other uracil nucleotides. Guanosine rapidly reversed both the reduction of guanine nucleotide pools and the elevation of cellular UTP contents. Enzymatic nucleotide analyses in cell and tissue extracts after treatment with ribavirin indicated that ribavirin 5'-triphosphate was an effective substrate for yeast hexokinase, yeast phosphoglycerate kinase, and nucleosidediphosphate kinase from yeast or bovine liver. These results were confirmed in detail by the use of synthetic ribavirin 5'-triphosphate and 5'-diphosphate. The latter nucleotide analog was also a substrate of pyruvate kinase from muscle. Mycophenolate-induced GTP deficiency was associated with an arrest of hepatoma cell growth in suspension culture. Ribavirin, at an equimolar concentration, was much less effective in this respect. None of the two inhibitors had a detectable effect, however, in vivo when guanine or uracil nucleotides were assayed in liver. This indicated that an inhibition of de novo guanylate synthesis in vivo can be compensated by salvage pathway synthesis.

Interactions between poly A and U been studied by spectrophotometric titrations. The dissociation constant of the groups in poly U having been determined at various ionic strengths, spectrophotometric titrations of solutions containing poly A and poly U in the ratio 1:1 and 1: 2 were performed in the alkaline pH range. By comparing the pK' of poly U with the transition pH of the (A+U) and (A+U+U) complexes, it was possible to calculate the free energy changes of the (A+U) and (A+U+U) helical structures. By doing so at various ionic strengths we were able to determine the electrostatic contribution to the free energy changes and to deduce the electrostatistic potential of the two and three stranded complexes. Titrations of the poly A-poly U aggregates have been performed at various temperatures and provided us with the free energy variation of these aggregations with temperature at various ionic strengths. From these results, it was possible to estimate enthalpy and entropy values of the formation of the (A+U) and (A+U+U) complexes. The energetic and thermodynamic relations of the conversion of the two- and the three stranded complex have been determined too.

A particulate enzyme preparation from dark grown mung bean shoots incorporates the glucosyl unit from UDP- (14C) -glucose into lipophilic products. These were identified by thin layer chromatography as a mixture of β-sitosterylglucoside and stigmasteryl-glucoside (80%) as well as the acylated forms of these substances (20%). The reaction is almost linear with time up to 10 minutes, optimal at pH values between 6.3 and 6.6 and does not require divalent cations. The acceptor sterols were shown to be present in the particulate preparation; exogenous sterols do not seem to act as acceptors. The same particulate enzyme preparation can also synthesize cellulose from UDPG 15 and lipid intermediates for its biosynthesis have been predicted 20. It appeared possible, therefore, that the sterylglucosides act as such intermediates. This, however, does not seem to be the case, since dilution of the UDP- (14C) -Glucose with unlabelled UDPG as well as its hydrolysis with phosphodiesterase within the course of an experiment demonstrates that the glucosyl units of the sterylglucosides show no turnover.

Während der Verfütterung von N-Nitroso-morpholin wurde der Nucleinsäure-Stoffwechsel in der Rattenleber studiert. 1. Schon kurze Zeit nach Beginn der Verabreichung von NM ist der Einbau von 14C-Orotsäure und 32P-Orthophosphat in die Kern-RNS der Leberzellen erhöht. 2. Das Basen-Verhältnis der RNS aus präcanzeröser Leber unterscheidet sich nur unwesentlich von dem der RNS aus normaler Leber. 3. Die Matrizen-Funktion der DNS, die zu verschiedenen Zeiten während der Carcinogenese aus der Leber isoliert wurde, erweist sich im RNS-Polymerase-System als unverändert.

The activity of UDPG-pyrophosphorylase [EC 2.7.7.9] was investigated during the morphogenesis (stalk and cap formation) of the unicellular and uninucleate green alga Acetabularia mediterranea. The activity of this enzyme is strongly related to the development of the cap. There was a substantial increase in the activity of the enzyme during cap formation. This increase in enzyme activity during cap formation also took place in anucleate cells. These findings are in good agreement with the fact that the glucose and galactose content of the cell wall of the cap is much higher than that of the stalk wall. The increase in enzyme activity in nucleate and anucleate cells was reversibly stopped by puromycin and actidion. Therefore the increase in the pyrophosphorylase activity must be due to synthesis of this enzyme. Actinomycin inhibits only the synthesis of the pyrophosphorylase in regenerating nucleate posterior parts of the cell. No inhibition occurs in anucleate cells with intact apical stalk because the cells contain a store of relatively stable messenger-RNA in the anterior parts of the stalk. On the other hand actinomycin inhibits the increase in chlorophyll content in both cell parts nucleate or anucleate. These findings indicate that the synthesis of UDPG-pyrophosphorylase in Acetabularia does not involve a direct regulatory function of nuclear or extranuclear genes (as for example chloroplast genes). Therefore we have to conclude that the synthesis of this enzyme was regulated in the cytoplasm at the level of translation.

Irradiation of uridylic acid (Up) in buffer (at 254 mµ or 280 mµ) yields the hydrated molecule (UpH2O), which on subsequent alkaline treatment decomposes to Up (65%) and a new compound A (35%) with the pyrimidine-ring opened. Irradiation in ice (254 mµ) leads to the formation of the uridylic acid dimer (Up/Up), which on re-irradiation reverts to Up (230 mµ, in neutral solution) or compound A, via Up and UpH2O) (254 mµ, in alkaline solution). Similarly the hydrated cytidylic acid (CpH2O) is formed by irradiation of cytidylic acid (Cp) in buffer (254 mµ). In a subsequent dark reaction most of the CpH2O-molecules revert to Cp, a minor part being deaminated to UpH2O. Alkaline treatment of an irradiated Cp-solution yields Cp, Up and compound A, the latter two arising by decomposition of UpH2O. Neither Cp nor Up dimers were found after irradiation of Cp in ice (254 mµ). All the photoproducts of Up and Cp, in which the pyrimidine-ring was still preserved, lost 1 - 3% of their phosphoric acid (Pi) under the experimental conditions. The alkaline decomposition of UpH2O is used as a method for the titration of hydrated uracil residues in irradiated ribonucleic acid (RNA). Following irradiation (254 mµ) the RNA is degraded by alkali completely to mononucleotides and three new photoproducts: compound A, ribose phosphate, and another compound B, which behaves like Up dimer. Form the amount of compound A found, the number of hydrated uracils originally formed can be calculated. Furthermore the kinetics of the formation of the photoproducts are studied. The biological implications of these studies are discussed.

A study on the effect of UV-irradiated polyuridylic acid on the incorporation of phenylalanine into the polypeptide precipitable through trichloroacetic acid, in a cell-free system from E. coli was made. Attempts were made to reactivate the UV-inactivated polyuridylic acid through hydrogen peroxide, uranyl acetate and visible light. We could show that polyuridylic acid irradiated at a dose of 1.2 ×105 ergs/mm2 could be completely reactivated, while the one irradiated at a higher dose of 2.4 ×105 ergs/mm2 could not be completely reactivated under the conditions of our experiment. We have studied the effects of hydrogen peroxide and uranyl acetate on UV-irradiated polyuridylic acid chemically as well. Our results altogether show that the photoreactivating effect of uranyl acetate and hydrogen peroxide is due to their ability to split the uracil dimers formed during UV-irradiation.