Characterization of Labeled Residues from 5-Diazouracil-2-C Incorporated into Ribonucleic Acids of Filamentous Escherichia coli

E. coli B, filamented with 5-diazouracil (DZU)-2-¹⁴C, yielded ribonucleic acid (RNA)-(DZU-2-¹⁴C) which was converted by pancreatic ribonuclease to ¹⁴C-mono-and oligo-nucleotides. The mixed ¹⁴C-mononucleotides isolated by diethylaminoethyl-cellulose fractionation were identified as cytidylic, uridylic, and hydroxyuridylic acids, by using a combination of paper chromatography and treatment with alkaline phosphatase and cytidine deaminase. Rifampin blocked incorporation of DZU-2-¹⁴C under conditions which inhibit RNA synthesis. Division inhibition by DZU-2-¹⁴C and the incorporation into Escherichia coli B were retarded by uracil but not by other RNA bases. In a pyrimidine-requiring E. coli, DZU substituted for uracil or cytosine to an extent limited by toxic effects. Cytosine and uracil retarded these effects and retarded the incorporation of DZU-2-¹⁴C into the pyrimidineless strain. A small proportion of DZU-2-¹⁴C was converted by the latter strain into hydroxyuridylic acid, but the bulk of the incorporated label was in cytidylic and uridylic acid, as in the wild strain.     

Growth-physiological changes in Escherichia coli and other bacteria during division inhibition by 5-diazouracil

Diazouracil (DZU) was shown to increase the mean cell volume of several bacterial species. The filaments which it induced with Escherichia coli B and Lactobacillus casei were examined in section by electron microscopy. In addition to the inhibition of division of E. coli by DZU, its effects were studied on mass increase, viability, and deoxyribonucleic acid, ribonucleic acid, and protein synthesis. The dependence of cell age on the division-inhibitory effect of DZU was examined in synchronous cultures. The division-inhibitory effect was not reversed by pantoyllactone, an antagonist of ultraviolet filamentation, or by l-tyrosine, which had been reported to antagonize DZU activity.     

Incorporation profiles of conjugated linoleic acid isomers in cell membranes and their positional distribution in phospholipids

Although the conjugated linoleic acids (CLA) have several isomer-specific biological effects including anti-carcinogenic and anti-adipogenic effects, their mechanisms of action remain unclear. To determine their potential effects on membrane structure and function, we studied the incorporation profiles of four CLA isomers (trans-10 cis-12 (A), trans-9 trans-11 (B), cis-9 trans-11 (C), and cis-9 cis-11 (D)) in CHO and HepG2 cells. All four isomers were incorporated into cellular lipids as efficiently as linoleic acid (LA), with the majority of the incorporated CLA present in membrane rafts. Of the four isomers, only CLA-A increased the cholesterol content of the raft fraction. Over 50% of the incorporated CLAs were recovered in phosphatidylcholine of CHO cells, but in HepG2 the neutral lipids contained the majority of CLA. The desaturation index (18:1/18:0 and 16:1/16:0) was reduced by CLA-A, but increased by CLA-B, the effects being apparent mostly in raft lipids. The Δ? desaturase activity was inhibited by CLAs A and C. Unlike LA, which was mostly found in the sn-2 position of phospholipids, most CLAs were also incorporated significantly into the sn-1 position in both cell types. These studies show that the incorporation profiles of CLA isomers differ significantly from that of LA, and this could lead to alterations in membrane function, especially in the raft-associated proteins.     

Photoaffinity labeling of the 1,25-dihydroxyvitamin D-3 receptor.

Underivatized 1,25-dihydroxy[26,27-3H]vitamin D-3 was successfully used to photoaffinity label the 1,25-dihydroxyvitamin D-3 receptor. The covalent incorporation of tritium into the receptor protein was induced by ultraviolet irradiation of the receptor-1,25-dihydroxy[26,27-3H]vitamin D-3 complex in crude pig intestinal nuclear extract. The amount of incorporated label increased with increasing time of irradiation and was dependent on light of wavelengths 220-280 nm. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography were used to demonstrate that label was incorporated primarily into the 1,25-dihydroxyvitamin D-3 receptor. In addition, the label incorporation was eliminated by competition with a 100-fold excess nonradioactive 1,25-dihydroxyvitamin D-3, indicating that the label was specific for the steroid binding site. Since 1,25-(OH)2[26,27-3H]vitamin D-3 is readily available and requires no special precautions for its preparation and handling, it should be a useful photoaffinity label for future studies of the receptor.     

Acetylcholine stimulates selective liberation and re-esterification of arachidonate and accumulation of inositol phosphates and glycerophosphoinositol in C62B glioma cells

Glioma C62B cells, incubated for 18 h with either an unsaturated (arachidonate or oleate) or saturated (palmitate or stearate) radioactive fatty acid, incorporated label into most species of cellular glycerolipids. Treatment of prelabeled C62B cells with 1 mM acetylcholine (ACh) resulted in an accumulation of radioactive phosphatidate irrespective of which fatty acid was used as a label. However, only in cells prelabeled with unsaturated fatty acids were increases in radioactive fatty acids observed. When exogenous radioactive arachidonate was added to C62B cells in the presence of 1 mM ACh, there was a rapid, selective, and transiently enhanced incorporation of label (several times the control) into phosphatidylinositol (PI). The ACh-enhanced incorporation into PI was not preceded by enhanced incorporation of label into sn-1,2-diacylglycerol or phosphatidate but was followed by an increased labeling of polyphosphoinositides. Similarly, incorporation of oleate into PI was enhanced by ACh. In contrast, ACh did not enhance the incorporation of label into any glycerolipids when saturated fatty acids were used. C62B cells, incubated with [2-3H]inositol for 18 h selectively incorporated label into phosphoinositides. Stimulation of [2-3H]inositol-labeled cells with 1 mM ACh in the presence of 25 mM LiCl resulted in a rapid accumulation of radioactive inositol phosphates (mono-, bis-, and trisphosphates) and glycerophosphoinositol. The accumulation of inositol trisphosphates preceded that of inositol monophosphate and glycerophosphoinositol, while the accumulation of glycerophosphoinositol paralleled the time required for the ACh-stimulated esterification of arachidonate. These results suggest that ACh stimulates activation of a phospholipase C in C62B cells and release of 1,4,5-inositol trisphosphate. There is subsequent activation of phospholipase A2, which in turn liberates arachidonate from PI. The resulting lyso PI is either rapidly reesterified with unsaturated fatty acid to resynthesize PI, or further deacylated to yield glycerophosphoinositol.     

Lipid biosynthesis by isolated plastids from greening pea, Pisum sativum

Isolated etioplasts from 8-day-old dark-grown pea seedlings incorporated [1-(14)C]acetate into lipid at a relatively low rate. Plastids from seedlings that had been illuminated for at least 2 hr showed an enhanced incorporation provided the plastids were illuminated during incubation with the labeled acetate. Dark incubation or the addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) decreased the acetate-incorporating activity of the developing chloroplasts to the level observed with etioplasts. Light had a marked effect on the type of fatty acid into which acetate was incorporated by the developing chloroplasts. Unsaturated fatty acids (mostly oleic acid) accounted for 60-80% of the incorporated label if the plastids were illuminated, but in the dark or in the presence of DCMU the unsaturated acids accounted for only 0-15% of the label incorporated into lipid. The effect of ATP on incorporation was dependent on the maturity of the chloroplasts; mature pea chloroplasts were inhibited by ATP, whereas in developing plastids there was a slight stimulation by ATP. Inhibition of acetate incorporation into lipid by DCMU appears to be due to inhibition of noncyclic phosphorylation. Incorporation was restored by reduced 2,3,5,6-tetramethylphenylenediamine, which restored phosphorylation, but not by reduced N,N,N',N'-tetramethylphenylenediamine.     

Effects of thymidine analogues on murine and human cells.

Three mouse tumour cell lines grew continuously in 3 micro M 5-bromodeoxyuridine (BUdR). One line (MC-2) produced a retrovirus and altered in morphology in the presence of BUdR or 5-iododeoxyuridine (IUdR). These effects, which could be reversed by growth in normal medium were similar to those reported for the B-16 mouse melanoma line. The B-16 line used in this study, however, as well as a variety of human cells (six melanoma lines and three fibroblast strains), were much more sensitive to BUdR, 0.03-0.1 micro M being the maximum tolerated levels for continuous growth. No virus production or changes in morphology were induced in these cells by BUdR, deoxyuridine (UdR), 5-fluorodeoxyuridine (FUdR) or thymidine (TdR). The results of cell labelling and growth studies showed a correlation of incorporation of BUdR into DNA with toxicity. Compared on a competitive basis with 1 micro M TdR, the order of incorporation of 1 micro M nucleosides by two human cell lines was TdR = BUdR = IUdR greater than UdR greater than FUdR. In contrast to previous reports that FUdR is incorporated into RNA but not into DNA, half of the FUdR label was found in alkalistable, DNase-sensitive material. Over 90% of the other compounds was incorporated into DNA. All of the UdR and 60% of the IUdR label was incorporated as thymidine; this conversion could be inhibited by labelling in the presence of FUdR.     

Direct evidence of the role of propionate in choline synthesis in rats

Wistar rats were injected with 2-14C-propionate in a dose of 30 mu Ci/100 g bw, 2 h after food intake. Two hours after isotope injection the rats were decapitated to determine specific radioactivity (SR) in liver and brain lipids, in liver phosphatidylcholine (PC) and its structural components. The label was incorporated in liver lipids in a far greater amount. In liver PC, SR appeared the highest in glycerin and less higher in the fraction of higher fatty acids. The least amount of the label from 2-14C-propionate was incorporated in choline. The fact of the label incorporation in choline was recorded for the first time.     

Phospholipid metabolism during regeneration of rat liver

The concentration and composition of phospholipids and mitotic activity in regenerating rat liver were studied. (1) The total amount of liver phospholipid increased approximately linearly during 48h after operation but without change in the relative concentrations of individual phospholipids. (2) The appearance of mitoses 30h after operation was accompanied by an increased incorporation of (32)P into the liver phospholipids. (3) The regenerating livers incorporated a higher percentage of the label into the phosphatidylserine+phosphatidylinositol fraction than those of control rats. The percentage of the label incorporated into phosphatidylethanolamine in these livers increased but decreased in the phosphatidylcholine.     

Studies on the Secretion of Maize Root Cap Slime: II. Localization of Slime Production

The distribution of fucose-containing polysaccharides in apical 1-cm sections of corn (Zea mays cv. SX-17) root tips was analyzed. Fucose-containing polysaccharides were localized predominantly in the apical 1 mm of the root, i.e., in the apical initials and root cap. An analysis of the distribution of incorporated radioactive label from l-fucose[(3)H] gave similar results. After a 2-hr incubation with fucose[(3)H], label was found principally in two components, namely a water-soluble slime fraction and hemicellulose. The incorporation of fucose into the water-soluble, ethanol-insoluble fraction was primarily in the apical 1 mm of the root, whereas incorporation into a water-insoluble, potassium hydroxide-soluble fraction was in the region 2 to 5 mm behind the root cap. Addition of sucrose to the incubation medium during fucose[(3)H] incorporation reduces label uptake but increases the amount of label in the fucose-rich secreted polysaccharide. The utility of fucose as a marker for the secreted polysaccharide was confirmed by demonstrating that no appreciable metabolism of this sugar occurs.     

Cell-Free Synthesis of Myelin Basic Proteins in Normal and Dysmyelinating Mutant Mice

Total polyribosomes were isolated from the brains of 16-20 day C57BL/6 mice, four neurological mutants (qk/qk, shi/shi, mld/mld, and jp/Y), and four heterozygote or littermate controls (qk/+, shil/+, mld, and jp littermates) and translated in a homologous, cell-free system. No differences were observed among the nine genotypes in either the yield of polysomes (32.2 +/- 0.6 A260/g brain) or in the incorporation of [35S]methionine into trichloroacetic acid-precipitable protein. However, when the four myelin basic proteins (BPs) were isolated from the translation mixtures little incorporation of [35S]methionine into the BPs was noted in those assays directed by polysomes from mld/mld or from shi/shi animals. Compared with C57BL/6 polysomes, mld littermate and shi/+ polysomes incorporated approximately half the levels of label into the four BPs while qk/+ and qk/qk incorporated normal and close-to-normal levels. Polysomes from jp littermates and jp/Y brains synthesized 66% and less than 15% of the levels of the 14K BP compared with C57BL/6 polysomes. Incorporation of label into the other three BPs was normal with jp littermate polysomes and about half the control levels with jp/Y polysomes. The data indicate that shi/shi and mld/mld mutants either produce altered BPs not recognized by our antibody or synthesize very low levels of BP. The data provide additional support for the notion that the qk/qk mutant synthesizes much higher levels of MBP than are incorporated into myelin. They also indicate that in the jimpy mutant the synthesis of the four BPs is affected to differing extents; thus, the mutant cannot be easily characterized as either an "assembly" or "synthesis" defect.     

Effects of the formaldehyde releasing preservatives dimethylol urea and diazolidinyl urea in several short-term genotoxicity tests

The two formaldehyde (FA)-releasers dimethylol urea (DMU) and diazolidinyl urea (DZU) are widely used as preservatives or additives. They were tested for genotoxicity in three short-term test systems, i.e. in the Salmonella typhimurium mutagenicity assay, in the in vitro micronucleus test with V79 Chinese hamster cells and in the in vitro tubulin assembly assay using isolated tubulin from pig brains. The polymerization products obtained in the tubulin assembly assay were examined additionally by electron microscopy.In the S. typhimurium mutagenicity assay with the pre-incubation assay both FA-releasers tested show a clear and concentration-dependent increase in the number of revertants in strains TA98, TA100 and TA102 with and without metabolic activation (rat liver S9 mix). In all cases, a biologically relevant increase in the number of revertants was achieved within the concentration range tested (DZU: 0.04-1.8 micromol per plate, DMU: 0.21-8.33 micromol per plate). FA was tested at 0.06-2.5 micromol per plate and lead to similar effects.Both compounds induce the formation of micronuclei (concentration range tested: DZU: 2.5-50 micromol/l, DMU: 3.3-333 micromol/l). However, DMU shows a comparatively weaker effect exclusively in the absence of the metabolizing enzymes. By contrast, DZU yields a distinct increase of the micronucleus rate in the absence and in the presence of S9. In addition, DZU predominantly causes an increase of large micronuclei, which suggests that this compound has a marked aneugenic potential. Cytotoxic effects accompany the clastogenic effects of both DMU and DZU.The examination of DMU and DZU in view of a possible aneugenic potential in the tubulin assembly assay yielded the following results: DMU at concentrations up to 10 mmol/l did not influence the formation of microtubuli, whereas DZU inhibited this process completely at 3 mmol/l. FA at 6 mmol/l completely inhibited the tubulin assembly. These results could clearly be confirmed by electron microscopy examination. The different potential of the two compounds with respect to the inhibition of tubulin formation is apparently due to a significant difference in the degree of FA release.According to these results, both compounds have to be considered as genotoxic in vitro. On account of these data and because of the widespread use of these two compounds in various products used in daily life, a reevaluation of the risk associated with these compounds seems to be necessary.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Arylisothiocyanate modification of sarcoplasmic Ca2+-stimulated ATPase

The related probes phenylisothiocyanate andp-sulfophenylisothiocyanate possess comparable reactivity with nucleophiles but are dissimilar in their solubility characteristics. The reagents are utilized to topologically characterize the sites of covalent interaction with the Ca²⁺-stimulated ATPase of sarcoplasmic reticulum membranes. The hydrophobic probe phenylisothiocyanate binds covalently to the membrane-integrated protein. The extent of covalent interaction of this probe is reduced to a limited level of label incorporation by either preincubation withp-sulfophenylisothiocyanate or by exposing the labeled protein to alkaline reductive conditions. With respect to the chemical nature a dual interaction of phenylisothiocyanate is postulated. Phenylisothiocyanate modifies the Ca²⁺-ATPase hydrophobically. In addition, aqueous-exposed nucleophiles (cysteine thiols) interact with both arylisothiocyanates. Inhibition of the Ca²⁺-stimulated ATPase activity is effected by either probe.     

Biosynthesis During Recovery of Heat-Injured Salmonella typhimurium

Salmonella typhimurium 7136 incorporated label from glucose-U-(14)C into nucleic acids, lipids, and pool material during recovery from heat injury. There was very little incorporation of label into protein during recovery.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Incorporation of coenzyme M into component C of methylcoenzyme M methylreductase during in vitro methanogenesis

Reduction of the methyl group of [methyl-3H,thio-35S]2-methylthioethanesulfonic acid to methane by a reconstituted enzyme system resulted in a slow incorporation of [thio-35S]2-mercaptoethanesulfonic acid (HS-CoM) into component C of the methylreductase system. Only 35S label was associated with component C. The ratio of incorporated HS-CoM to component C was 1.96 to 1. The ratio of HS-CoM to factor F430, the nickel-containing cofactor of component C, was 1.18 to 1. Extraction of factor F430 from the protein resulted in the release of 62 +/- 8% of the 35S label, but the label was not covalently bound to F430. The incorporation of label into component C was coupled to methyl group reduction; no label was found associated with component C from a reconstituted reaction containing unlabeled 2-methylthioethanesulfonic acid and [thio-35S]HS-CoM.     

In vitro alanine utilization by rat interscapular brown adipose tissue

The in vitro oxidation to CO2 and tissue incorporation of alanine label by pieces of rat interscapular brown adipose tissue (IBAT) has been investigated. Insulin increased both uptake and oxidation of alanine, as well as the incorporation of alanine label into tissue. This effect only was observed in the presence of glucose in the incubation medium. Noradrenaline hampered alanine incorporation, not affecting its rate of oxidation. IBAT from 4-h cold-exposed rats showed a higher alanine utilization than that of controls; however, IBAT pieces from both 36-h starved and 30-day cold-exposed rats presented lower rates of alanine utilization. The main fate of alanine taken up by the IBAT pieces was its oxidation to CO2. Part of the label was also incorporated into the fatty acid fraction of lipids. The results obtained in this study agree with a possible role of alanine as alternative energetic substrate for IBAT.     

Biosynthesis of caldariellaquinone in Sulfolobus spp.

The biosynthesis of caldariellaquionone (CQ) was studied in species of Sulfolobus by measuring the incorporation of stable isotopically labeled tyrosines into CQ. By feeding a series of tyrosines labeled with deuterium or 13C and then measuring the extent and position at which label was incorporated into CQ by mass spectrometry, it was shown that more than 95% of the label was incorporated into the benzo[b]thiophen-4,7-quinone moiety of CQ. From the labeling experiments, it is concluded that the benzo[b]thiophen-4,7-quinone is derived as an intact unit from all of the carbons of tyrosine except C-1.     

Incorporation of polyunsaturated fatty acids into bis(monoacylglycero)phosphate and other lipids of macrophages and of fibroblasts from control and Niemann-Pick patients

On the basis of earlier studies of rabbit pulmonary alveolar macrophages, the incorporation of 14C-labelled polyunsaturated fatty acids into the lipids of human fibroblasts from patients with various phenotypes of Niemann-Pick disease was examined in order to define further the disturbance in metabolism of bis(monoacylglycero)phosphate occurring in these disorders. Docosahexaenoic acid, which had not been studied previously, was found to be incorporated by macrophages into bis(monoacylglycero)phosphate in a highly selective fashion and was therefore used along with arachidonic acid for studies of fibroblasts. Following incubation of fibroblasts in serum-free medium for 60 min, the distribution of arachidonic acid label in lipids was: phosphatidylcholine, 51%; phosphatidylethanolamine, 12%; phosphatidylinositol, 9.5%; and bis(monoacylglycero)phosphate, 2.3%; and of docosahexaenoic acid label was 36, 20, 2.6 and 10.3% respectively. Phosphatidylinositol had the highest specific activity of arachidonic acid label and bis(monoacylglycero)phosphate of docosahexaenoic acid label. Prolongation of incubation to 21 h, with or without removal of label remaining in the medium at 1 h, resulted in proportional redistributions with phosphatidylcholine decreasing and phosphatidylethanolamine increasing. In bis(monoacylglycero)phosphate and phosphatidylinositol, the proportions of arachidonic acid label decreased and increased respectively, whereas the proportions of docosahexaenoic acid label in these lipids were unchanged. As virtually all label taken up by cells was esterified, these redistributions are taken to reflect transacylations. In Niemann-Pick cells, the expected redistribution of arachidonic acid label in bis(monoacylglycero)phosphate failed to occur with cell types A and B which are deficient in sphingomyelinase-phospholipase C, and excess label accumulated after a 21-h incubation. Excess docosahexaenoic acid label also accumulated in the bis(monoacylglycero)phosphate of these cells. The highly selective incorporation of docosahexaenoic acid in two cell types suggests a special role for bis(monoacylglycero)phosphate in the metabolism of n-3 polyunsaturated fatty acids. A high specific activity found early in incubations of macrophages suggests that polyunsaturated fatty acids may be incorporated into phospholipids during de novo synthesis of phosphatidic acid.