Selective recognition in erythrophagocytosis by mouse peritoneal macrophages. Isolation and purification of a possible self-marker from mouse erythrocyte membranes

The percental participation of exogenous cytidine in liver RNA synthesis was determined after application of ³H-cytidine to rats. The amount of exogenous cytidine was varied by a factor of 5 × 10⁵, between 0.000 02 and 10.0 μg/g rat. With the ³H-cytidine doses and specific activities most frequently reported in the literature, the percental participation of the exogenous precursor is only about 0.1%, with 99.9% of the cytidylic acid incorporated into RNA under these conditions being of endogenous origin.The results show that the upper limit of the tracer dose of exogenous cytidine is about 1.0 μg/g rat. Within this tracer region 1.8% of ³H-activity—and therefore 1.8% of the amount of exogenous cytidine—is incorporated into liver RNA. The dependence of the percental participation on the duration of the experiments is examined.It is shown that autoradiographic grain density and specific activity of RNA can only be regarded as direct measures for the rate of RNA synthesis in different cells and animals if the percental participation of exogenous cytidine in RNA synthesis is generally of equal value.Comparable situations exist in the incorporation of ³H-thymidine into DNA as shown by earlier experimental work.     

Metabolism of [6-14C]orotate by shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus

Incorporation of radioactivity from [6-¹⁴C]orotate into the pyrimidine constituents of shoots of Pisum sativum, Phaseolus vulgaris and Lathyrus tingitanus was examined with special reference to the unusual pyrimidine constituents. With each species, although 80% of the orotate supplied was catabolized to β-alanine, all the pyrimidine derivatives became radioactively labelled. With Pisum, the major part of the radioactivity incorporated into pyrimidines was located in UMP and the uracil derivatives, including the uracilyl amino acids willardiine and isowillardiine. With Phaseolus, UMP and the uracil derivatives were again the major radioactive products; incorporation of radioactivity into 5-ribosyluracil (pseudouridine), which accumulates in Phaseolus tissues, was comparable to the incorporation into orotidine and twice that found in cytidine. Lathyrus incorporated a substantially larger part of the presented [6-¹⁴C] orotate into pyrimidine derivatives than did the other two species. CMP was the most highly radioactive product, followed next by lathyrine and UMP. Surprisingly, 20% of the total radioactivity incorporated into pyrimidines by Lathyrus was located in the pyrimidine amino acid lathyrine. This confirms previous evidence that lathyrine is essentially a product of the orotate pathway. The overall recovery of radioactivity in all three species was 93–95%. The data emphasize the necessity of including the less common pyrimidine constituents, as well as the common ones, in quantitative studies of pyrimidine metabolism in plants.     

Characteristics of a phosphatidylinositol exchange activity of soybean microsomes

Microsome fractions from hypocotyls of dark-grown soybean (Glycine max [L.] Merrill) seedlings incorporated myo-inositol into phosphatidylinositol by an exchange reaction stimulated by Mn²⁺ (optimum at 10 mm) and cytidine nucleotides (CMP = CDP CTP) but not by Mg²⁺ or nucleotides other than cytidine nucleotides. The activity was membrane associated, with an optimum pH of 8, stimulated by auxin, and inhibited by certain thiol reagents or by heating above 40°C. With radioactive inositol, phosphatidylinositol was the only radioactive product. That turnover was by myo-inositol exchange was verified from experiments where unlabeled inositol replaced already incorporated inositol with approximately the same kinetics as for the incorporation of label. Both the incorporation and the displacement reactions were stimulated by Mn²⁺ and CMP and both were responsive to auxin with comparable dose dependency. Corresponding exchange activities with choline or ethanolamine were not observed. The phosphatidylinositol-myo-inositol exchange activity was low or absent from plasma membrane, tonoplast, and mitochondria enriched fractions. The activity co-localized on free-flow electrophoresis and aqueous two-phase partition with NADPH cytochrome c reductase and latent IDPase, markers for endoplasmic reticulum and Golgi apparatus, respectively. With microsomes incubated with both ATP and inositol, polyphosphoinositides were unlabeled demonstrating separate locations for the inositol exchange and phosphatidylinositol kinase reactions. Thus, the auxin-responsive inositol turnover activity of soybean membranes is distinct from the usual de novo biosynthetic pathway. It is not the result of a traditional D-type phospholipase and appears not to involve plasma membrane-associated polyphosphoinositide metabolism. It most closely resembles previously described phosphatidylinositol-myo-inositol exchange activities of plant and animal endoplasmic reticulum.     

Autoradiography ofCoelopa frigida (Diptera) embryos,after labeling with3H-uridine during various phases of oogenesis

Summary The distribution of tritiated (³H) uridine or its derivatives in embryos of the kelp flyCoelopa frigida is analyzed by autoradiography. The radioactive label is introduced into the growing oocyte, after injectio into females at different time periods post eclosion. The majority of the label remains in the cytoplasm for the entire embryonic development and is presumably incorporated into stable RNA. Only when the precursor is injected toward the final stages of oogenesis, can presence of label be detected in the nuclei, for a short time period during the blastodern stage. Since no preferential location in nucleoli is found, it appears possible that labeled cytidine could have been derived from the original precursor which would be available for incorporation into DNA.     

Metabolism of cytidine and uridine in bean leaves

The metabolism of cytidine-2-¹⁴C and uridine-2-¹⁴C was studied in discs cut from leaflets of bean plants (Phaseolus vulgaris L.). Cytidine was degraded to carbon dioxide and incorporated into RNA at about the same rates as was uridine. Both nucleosides were converted into the same soluble nucleotides, principally uridine diphosphate glucose, suggesting that cytidine was rapidly deaminated to uridine and then metabolized along the same pathways. However, cytidine was converted to cytidine diphosphate and cytidine triphosphate more effectively than was uridine. Cytidine also was converted into cytidylic acid of RNA much more extensively and into RNA uridylic acid less extensively than was uridine. Azaserine, an antagonist of reactions involving glutamine (including the conversion of uridine triphosphate to cytidine triphosphate), inhibited the conversion of cytidine into RNA uridylic acid with less effect on its incorporation into cytidylic acid. On the other hand, it inhibited the conversion of orotic acid into RNA cytidylic acid much more than into uridylic acid. The results suggest that cytidine is in part metabolized by direct conversion to uridine and in part by conversion to cytidine triphosphate through reactions not involving uridine nucleotides.     

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

The maximal stoichiometry for [³H]GTP binding to depolymerized tubulin with saturating amounts of added [³H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [³H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.     

Metabolism of pyrimidine bases and nucleosides in Neisseria meningitidis.

In Neisseria meningitidis, uridine, deoxyuridine, cytosine, cytidine, or deoxycytidine could not be used by uracil-requiring mutants as pyrimidine sources. Consistent with these findings, only 5-fluorouracil of the different fluoropyrimidine bases and nucleosides showed any inhibitory effect on the growth of four prototrophic strains of N. meningitidis. Likewise, only radioactive uracil was readily incorporated into nucleic acids, whereas uptake of radioactive uridine, cytosine, or cytidine could not be demonstrated. Uracil was converted to uridine 5'-monophosphate by uracil phosphoribosyltransferase, whereas enzyme activities for conversion of cytosine or any of the nucleosides were not detectable in meningococcal extracts.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

Studies on Deoxyribonucleotide Synthesis in Vicia faba

The incorporation of uniformly labelled [¹⁴C] cytidine into the nucleic acids was studied in root tips of Vicia faba. Cytidine was found to be incorporated into RNA and DNA and the specific activities of the individual mononucleotides were deter- mined. The pyrimidine nucleotides were degrade and the ratio between the specific activity of the pentose and the specific activity of the base was determined for each nucleotide. CMP of RNA and deoxy CMP of DNA bad almost the same pentose: base ratios as The cytidine added to the incubation medium. It was concluded that the administered cytidine or a derivative of it was reduced to the corresponding deoxycytidine compound without breakage of the bond between pentose and base. [¹⁴C)-cytidine was transformed to UMP of RNA with some loss of radioactivity from the pentose and had almost the same pentose: base ratio as deoxy TMP of DNA. This indicates that the formation of thymidine phosphates involved The reduction of a uridine compound. Furthermore the incorporation of ¹⁴C-labelled thymidine, deoxyadenosine and deoxyguanosine into DNA was studied. Deoxyguanosine was found to be incorporated only to a slight extent. This finding has been discussed in relation to previous results.     

The fate of the larval storage protein calliphorin during adult development of Calliphora vicina

Purified calliphorin labelled with [¹⁴C]phenylalanine by in vivo synthesis was injected into mature Calliphora vicina larvae to examine the distribution of label during metamorphosis and in young adults. About 2.5% of the administered radioactivity was expired during adult development, and 10% was incorporated in the puparium. At the middle of adult development calliphorin was the only detectable radioactive soluble protein in contrast to the situation in the teneral adult where most soluble proteins were labelled. All organs and/or tissues of 4-day flies were radioactive; nearly half the phenylalanine of calliphorin was incorporated into flight muscle, actin and myosin being strongly labelled. Earlier hypotheses that calliphorin is a larval storage protein are now experimentally verified.     

Phosphatidylinositol synthesis by a mn-dependent exchange enzyme in castor bean endosperm

myo-Inositol is incorporated into phosphatidylinositol by an exchange reaction associated with the endoplasmic reticulum fraction isolated from post-germination castor bean endosperm. The reaction requires Mn²⁺, has a pH optimum of 8.0, an apparent K_m for myo-inositol of 26 micromolar, and is stimulated about 15-fold by certain cytidine derivatives. The cytidine derivatives appear to be converted to CMP, which may be the only active stimulator. These optimal exchange reaction conditions, both with and without CMP, differ from those for cytidine-5′ -diphosphodiglyceride: myo-inositol transferase (EC 2.7.8), so the exchange does not appear to be a reversal of the transferase. This conclusion is augmented by the low rates of CDP-diglyceride formation from cytidine derivatives when compared to the high rate of myo-inositol incorporation into phosphatidylinositol in the presence of the same cytidine derivatives and identical reaction conditions.     

Plasmodium falciparum: Synthesis of glycoprotein by cultured erythrocytic stages

The human malarial parasite, Plasmodium falciparum, incorporated significant radioactivity into glycoconjugates when cultured in the presence of [¹⁴C]- or [³H]glucosamine for 48 to 50 hr. Digestion of the labeled proteins with pronase and subsequent precipitation with absolute ethanol showed that 90 to 95% of the radioactive glucosamine was incorporated into the precipitated material. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the labeled macromolecules revealed eight bands with approximate molecular weights from 19,000 to 90,000 daltons.     

Intracellular formation of analogues of cyclic AMP: Studies with brain slices labeled with radioactive derivatives of adenine and adenosine

A variety of radioactive analogs of adenine and adenosine were incubated with guinea pig cerebral cortical slices. Neither 1,N⁶-ethano[¹⁴C]adenosine nor 1,N⁶-ethanol[¹⁴C]adenine were significantly incorporated into intracellular nucleotides. 2-chloro[8-³H]adenine was incorporated, but at a very low rate and conclusive evidence for the formation of intracellular radioactive 2-chlorocyclic AMP was not obtained. N⁶-Benzyl[¹⁴C]adenosine was converted only to intracellular monophosphates and significant formation of radioactive N⁶-benzylcyclic AMP was not detected during a subsequent incubation. 2′-Deoxy-[8-¹⁴C] adenosine was converted to both intracellular radioactive 2′-deoxyadenine nucleotides and radioactive adenine nucleotides. Stimulation of these labeled slices with a variety of agents resulted in formation of both radioactive 2′-deoxycyclic AMP and cyclic AMP. Investigation of the effect of various other compounds on uptake of adenine or adenosine suggested that certain other adenosine analogs might serve as precursors of abnormal cyclic nucleotides in intact cells.     

Incorporation of Radioactive Precursors into DNA and RNA of Plasmodium knowlesi in vitro

SYNOPSIS. Cultures of the intra-erythrocytic stages of Plasmodium knowlesi incubated in vitro utilized all the pre-formed radioactive purines tested (adenine, adenosine, deoxvadenosine, guanine, guanosine and hypoxanthine) but none of the pyrimidines (thymine, thymidine, uracil, uridine, cytidine and deoxycytidine). They did, however, utilize the pyrimidine precursor orotic acid.
All precursors analysed, including deoxyadenosine, were incorporated into both DNA and RNA (in the ratio of ∼1:3) but 19% was incorporated into other unidentified compounds. ³Hadenosine was incorporated into adenine and guanine residues of both DNA and RNA.
No unambiguous evidence was obtained for any periodicity in the synthesis of DNA or RNA in our cultures, even tho cultures remained as synchronous in vitro as they are in vivo. An estimate is presented of the amount of DNA made during one cycle in vitro.     

Catalysis of accurate poly(C)-directed synthesis of 3'-5'-linked oligoguanylates by Zn2+

Zn²⁺ is an efficient catalyst for the oligomerization of guanosine 5′-phosphorimidazolide on a polycytidylic acid template. Up to 75% of the input ImpG² is converted to oligomers with a mean chain length up to 10. Material longer than (pG)₃₀ can be detected. The oligomeric products are predominantly 3′-5′-linked.If poly(C) is incubated with ImpG and an equimolar quantity of the 5′-phosphorimidazolides of adenosine, uridine or cytidine, in the presence of Zn²⁺, ImpG is incorporated at least 200 times more efficiently than “incorrect” nucleotides.     

Selective incorporation of 14C-arachidonic acid into the phospholipids of intact tissues and subsequent metabolism to 14C-prostaglandins

A method is described for the efficient incorporation of radioactive arachidonic acid into the lipids of rabbit hearts and kidneys. Infusion of ¹⁴C-arachidonate through perfused tissues resulted in the quantitative removel of label from the media. Analysis of the lipids from tissues labeled by this procedure revealed that the majority of the ¹⁴C-arachidonate was incorporated into phospholipids. Essentially all of the radioactivity in phosphatidylcholine was found in the 2-position. Subsequent to the ¹⁴C-arachidonate infusion, stimulation of prostaglandin biosynthesis (e.g. by bradykinin) resulted in the release of radioactive prostaglandins. This suggests that the ¹⁴C-arachidonate is incorporated in a manner such that it is available for homone-stimulated prostaglandin biosynthesis. The method described allows both qualitative and quantitative analysis of arachidonate metabolism in intact tissues and offers significant advantages over other presently used methods.     

Conformation of 2-aminofluorene-modified DNA

Minimized potential-energy calculations were performed to determine the conformation of the 2-aminofluorene (AF) adduct to dCpdG at guanine C-8. The AF adduct has many low-energy conformers in both the anti and the syn domains of the guanine. This is in contrast with the acetylated adduct, (AAF), which greatly prefers the syn domain. Two types of low-energy guanine anti-conformations were obtained: (1) conformers that preserve guanine–cytidine stacking and (2) conformers with fluorene–cytidine stacking. Of special importance are conformers with ω′,ω,ψ = g^?, g^?, g⁺, characteristic of normal A- or B-helices, which are found in both groups. No conformers of this type were obtained for the acetylated AAF adduct. The guanine–cytidine stacked from with this conformation can be incorporated in the B-helix without any distortion, with the carcinogen situated at the helix exterior. The fluorene in this model can slide into the helix to yield a fluorene–cytidine stacked minimum-energy conformation. This requires no denaturation, although one base pair is unstacked and the helix axis is bent. Low-energy syn-conformations, similar to those obtained for the AAF adduct, were also computed. These were either guanine-cytidine stacked or fluorene–cytidine stacked. The syn froms are less likely to be observed in larger DNA polymers of the adduct, since they cause more distortion than the anti-conformations. However, they might well be observed in crystals of small subunits, and they should contribute significantly to the population in solution.     

Involvement of dolicholmonophosphate in the formation of specific mannosyl-linkages in yeast glycoproteins

A membrane fraction from Saccharomyces cerevisiae catalyzes the transfer of mannosyl residues from GDP-Man partly via dolicholmonophosphate into a heterogenous glycoprotein fraction. The pattern of radioactive products obtained after mannosylation with GDP-[¹⁴C]Man is similar to that obtained with dolicholmonophosphate-[¹⁴C]mannose. In each case more than 70% of the radioactivity can be released by β-elimination. Evidence is presented, that only the mannosyl residue directly linked to protein is incorporated via dolicholmonophosphate.     

Efficient catalysis of polycytidylic acid-directed oligoguanylate formation by Pb2+

Pb²⁺ is a very effective catalyst for the template-directed self-condensation of guanosine 5′-phosphorimidazolide on a polycytidylate template. In the presence of 0.01 m-Pb²⁺ at 0 °C, up to 85% of the input ImpG ² is converted to a mixture of oligomers with a mean chain length of up to 17. Substantial amounts of material longer than (pG)₄₀ are formed. About half the ImpG reacts within an hour. The oligomeric products are predominantly (2′–5′)-linked.If poly(C) is incubated with ImpG and an equimolar quantity of the corresponding derivative of adenosine, uridine or cytidine, the “incorrect” nucleotide, is incorporated about 10% as often as G.     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.