Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

Utilization of nucleoside diphosphate glucoses in developing cotton fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-¹⁴C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-¹⁴C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.     

Studies on the biochemistry and fine structure of silica-shell formation in diatoms

Summary The distribution of radioactivity in ethanol-water-soluble compounds after short periods of photosynthetic incorporation of ¹⁴CO₂ is consistent with the operation of the photosynthetic carbon reduction (PCR) cycle in the fresh water diatom Navicula pelliculosa. Incorporation of ¹⁴CO₂ for extended time periods established the presence of the intermediates of the PCR and tricarboxylic acid (TCA) cycles, amino acids, and organic acids; free sugars were not observed. The main labelled soluble carbohydrate was a glucan. Hydrolysis of the radioactive insoluble material indicated the presence of carbohydrates containing several distinct sugars, and proteins with the usual amino-acid composition. During silicon starvation of exponentially growing cultures, rates of incorporation of both ³²P _i and ¹⁴CO₂ decreased. Incorporation into the lipid increased, with a corresponding decrease into protein and carbohydrate. Reintroduction of Si to staryed cells led to an increased rate of incorporation of both isotopes, and transient changes in the radioactivity in most metabolic intermediates investigated. After 30 min the radioactivity in all PCR cycle intermediates, except phosphoglyceric acid, had increased by about 300%. The radioactivity of citrate and -keto-glutarate increased, whereas that of other TCA-cycle intermediates decreased. An initial decrease in the levels of glutamate, aspartate and glutamine was apparently reversed by cleavage of glutamate-aspartate peptides, as radioactivity of other amino acids increased. Incorporation into the soluble glucan and into protein increased markedly although the rate of incorporation into insoluble carbohydrates remained constant.     

Metabolic regulation in the senescing tobacco leaf: I. Changes in pattern of p incorporation into leaf disc metabolites

Changes in phosphate metabolism were explored in discs from tobacco (Nicotiana tabacum) leaves of three contrasting types: green leaves which were fully expanded and attached to the plant, leaves which had yellowed following excision and dark starvation, and leaves which had yellowed while attached to the plant. 2,4-Dinitrophenol at 10⁻⁵m stimulated the respiration rate of discs from green and yellow-detached leaves only slightly, but markedly stimulated that of discs from yellow-attached leaves. Following a 10-minute uptake period the incorporation of ³²P-orthophosphate into phosphate esters and lipids of discs from yellow-detached leaves was resistant to 2,4-dinitrophenol, whereas in discs from green and yellow-attached leaves it was inhibited by 2,4-dinitrophenol. Incorporation into a salt-soluble fraction containing unidentified nucleotide material showed converse behavior in that it was stimulated by 2,4-dinitrophenol in discs from green and yellow-attached leaves; in discs from yellow-detached leaves it was resistant to 2,4-dinitrophenol. In discs from yellow-detached and yellow-attached leaves there was a shift in the labeling pattern of phosphate esters toward increased label in hexose phosphates at the expense of adenine nucleotides, 3-phosphoglycerate, and phosphoenolpyruvate. It is concluded that incorporation into phosphate esters in discs from yellow-detached leaves is by substrate level phosphorylation coupled to enhanced aerobic glycolysis. In discs from yellow-attached leaves, on the other hand, incorporation depends on oxidation phosphorylation, and it is suggested that the shift in labeling pattern is caused by senescence-induced changes in activity of glycolytic enzymes.     

Effect of carbon tetrachloride administration on the synthesis of triglycerides and phospholipids in rat liver

In vivo incorporation of choline-methyl-(14)C into liver lecithin and its biosynthetic precursors was studied in CCl(4)-treated rats. Radioactivity in cytidine diphosphoryl (CDP-)choline and lecithin was reduced to one-third of control levels, whereas that of phosphorylcholine was increased to 4.7 times control levels. Incorporation of phosphorylcholine-(32)P into lecithin by homogenates prepared from livers of CCl(4)-treated animals was reduced, but conversion of CDP-choline-(32)P to lecithin by the isolated microsomal fraction did not show any significant depression. A block in the synthesis of CDP-choline is indicated. The in vivo utilization of methionine for lecithin synthesis was not affected. After intravenous injection of palmitic acid-1-(14)C, radioactivity of triglycerides from microsomal and mitochondrial fractions was markedly lower than the controls, whereas radioactivity of triglycerides in the soluble fraction was greatly increased. Radioactivity of diglycerides changed from 0.5% of total lipids in the control to 10% of total lipids in CCl(4)-treated animals. Incorporation of palmitic acid into phospholipids was also suppressed. The results demonstrate that synthesis of both phospholipids and triglycerides is inhibited in rats 4-5 hr after CCl(4) administration.     

Effects of inhibitors of lipid synthesis on the replication of Rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins.

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholestetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1(-14C)]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [35S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examinatin of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Effects of inhibitors of lipid synthesis on the replication of rous sarcoma virus. A specific effect of cerulenin on the processing of major non-glycosylated viral structural proteins

The effects of two inhibitors of lipid biosynthesis on the replication of Rous sarcoma virus Prague C strain in chick embryo fibroblasts have been examined in media containing delipidated serum. 25-Hydroxycholesterol, which markedly inhibits the incorporation of [1-¹⁴C]acetate into sterols, had no effect on the formation of infectious virions or on the synthesis and processing of intracellular virion proteins. Cerulenin strongly inhibited [1-¹⁴C]acetate incorporation into fatty acids and partially inhibited its incorporation into sterols in chick embryo cells. Rous sarcoma virus production as measured by focus formation and by the production of [³⁵S]methionine-labeled virions was strongly inhibited within 5 h after cerulenin addition to infected cultures. Examination of extracts of these cells revealed the accumulation of the 76 000 dalton precursor (Pr76) of the major non-glycosylated virion structural proteins, p27, p19, p15 and p12. The failure to process the 76 000 dalton precursor was coincident in time with the decrease in viron production. Neither whole serum nor mixtures of fatty acids plus cholesterol were able to reverse the effects of cerulenin.     

Incorporation of 2-14C-acetate into isolated nuclei and cytosolic lipids of rat thymus cells

It is generally recognized nowadays that active lipid metabolism takes place in the nucleus of a mammalian cell. Experimental data testify to the biosynthesis of polyphosphoinositides and phosphatidylcholine and reveal corresponding enzymes within nuclei of mammalian cells. These findings suggest that lipidmediated signaling pathways in nuclei operate independently of lipid-mediated regulatory mechanisms functioning in membranes and cytosol. To explore the pathways of intranuclear lipid biosynthesis, we studied incorporation of 2-¹⁴C-acetate into lipids of cytosol and isolated nuclei of rat thymus cells after separate and combined incubation with the labeled precursor. The most efficient incorporation of 2-¹⁴C-acetate into lipids (cholesterol, free fatty acids, and phospholipids) was observed in a reaction mixture containing cytosol. When the reaction mixture contained only nuclei, incorporation of the radioactive precursor into lipids also took place, but specific radioactivity of the lipids was essentially lower than in the cytosol. In both cases, 2-¹⁴C-acetate incorporated into phosphatidylethanolamine, sphingomyelin, phosphatidylserine, phosphatidylinositol, and cardiolipin. Phosphatidylcholine, the most abundant membrane phospholipid, demonstrated the lowest radioactivity, which was significantly lower than that of phosphatidylethanolamine. Incorporation of newly synthesized free fatty acids in nuclear phospholipids was inhibited, if nuclei were incubated with cytosol. As a result, radioactive free fatty acids were accumulated in nuclei, while in cytosol they were efficiently incorporated into phospholipids. The levels of phospholipids and cholesterol remained constant regardless of incubation protocol, while the overall yield of free fatty acids decreased after combined incubation of nuclear and cytosolic fractions or after incubation of cytosol without nuclei. Putative mechanisms underlying the appearance of radioactive lipids in isolated nuclei of thymus cells are discussed.     

Effects of prostaglandins and luteinizing hormone-releasing hormone on phosphatidic acid-phosphatidylinositol labeling in rat granulosa cells

In cultures of rat granulosa cells, luteinizing hormone-releasing hormone (LHRH) increases 32P incorporation into both phosphatidylinositol (PI) and phosphatidic acid (PA). After 20 min, the level of radioactivity was three- to four-fold (p less than 0.01) above control in the PI and PA fractions, respectively. The stimulatory effect of LHRH on 32P incorporation was limited to PI and PA. Similar to the effects of LHRH, a rapid and marked increase of 32P incorporation into both PI and PA is observed upon addition of prostaglandin F2 alpha (PGF2 alpha) (10(-5)M) to rat granulosa cells. Incorporation of radioactivity into PA was already increased (p less than 0.05) by 2 min following PGF2 alpha addition, while the increase in 32P-labeled PI became significant (p less than 0.01) by 5 min. In contrast to PGF2 alpha, the labeling of PI and PA following the addition of PGE2 (10(-5)M) was not significantly different from control levels during the entire 10 min of incubation. The sensitivity of the increased PA-PI labeling induced by LHRH and PGF2 alpha is compared in another experiment. After 20 min incubation 10(-6)M LHRH increased PI and PA labeling by six- and four-fold, respectively. Although the effect of PGF2 alpha is less than that of LHRH, 10(-5)M PGF2 alpha significantly (p less than 0.01) increased PI and PA labeling by three- and two-fold, respectively. By contrast, 10(-6)M PGE2 failed to affect 32P incorporation into the various phospholipid fractions, but a small enhancement (p less than 0.05) of PI and PA labeling was observed only at 10(-5)M PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on ectoderm and mesoderm of mouse limb buds.

The effects of cytosine arabinoside, 6-aminonicotinamide, and 6-mercaptopurine riboside on the incorporation of [14C] glucose moieties and [32P] phosphate into acid-soluble material and lipids, RNA, DNA, and protein were measured in the dissected mesoderm and ectoderm of mouse limb buds at the 42-45 (day 11) somite stage. Due to the different proliferative capacities of the two tissues the incorporation of the precursors into mesodermal cells was considerably higher the than into ectodermal ones. Cytosine arabinoside inhibited the incorporation of the precursor moieties only into DNA, but very early after its application. This effect was more obvious in mesoderm than ectoderm. 6-Aminonicotinamide interfered only with glucose metabolism, whereas the incorporation of phosphate was not affected. 14C radioactivity in the various cell components was similarly reduced in mesoderm and ectoderm. 6-mercaptopurine riboside caused an increased incorporation of precursor material in all fractions studied in the mesoderm as well as in the ectoderm during the first 12 hours. This was succeeded by a dramatic decrease of incorporated 14C and 32P radioactivity. Differences of response in the tissues could not be detected with this drug. It is suggested that the malformations of the extrmities caused by these antimetabolites may be predominantly attributed to changes in the cell function rather than to gross effects on cell metabolism.     

Inhibition of oleic acid incorporation into a non-lipid fraction by chloroacetamide herbicides

Oleic acid is incorporated into an insoluble fraction left over after lipid extraction in Scenedesmus acutus. This incorporation is extremely sensitive to the chloroacetamide herbicide, metazachlor (I₅₀= ca 20 nM). Therefore, factors influencing the incorporation of radioactivity from oleic acid into this non-lipid fraction were investigated. S. acutus cells were cultivated under various conditions with or without inhibitors and [¹⁴C]-oleic acid was supplied to the algae; the lipids were extracted and the radioactivity incorporated in the remaining fraction monitored. The inhibition seemed specific for chloroacetamides and related classes since it was also observed with alachlor, dimethenamid and mefenacet (an oxyacetamide). In contrast, it could not be found with diuron, oryzalin, nor could it be observed with a non-herbicidal metazachlor derivative or iodoacetamide. Incorporation of oleic acid into that fraction required meta-bolically active cells and was stimulated by light. Other fatty acids (16:0, 18:2, and 18:3) were also incorporated into the non-lipid fraction but their incorporation was not inhibited by metazachlor. Among other components, the fraction contains proteins. However, a possible specific effect of chloroacetamides on the binding of oleic acid to proteins or on the in vitro activity of lipid transfer proteins could not be detected. Not much is known yet about mechanism and chemistry of oleic acid incorporation but this finding opens a new path for investigations towards the primary target of these herbicides.     

Isolated Coxiella burnetii synthesizes DNA during acid activation in the absence of host cells

Two populations of Coxiella burnetii were isolated from fibroblast tissue cultures and examined for their ability to synthesize DNA when incubated in a defined medium. Both the populations released by mechanical lysis of heavily infected host cells, as well as those recovered from the tissue culture medium, incorporated H3 32PO4 into DNA. Incorporation occurred at pH 4.5 but not at pH 7.0, and proceeded for 12-15 h. When incorporation of [3H]thymidine was studied, only the organisms obtained by mechanical lysis of host cells were active. Those which had been released by natural means into the tissue culture medium, and then recovered for study, did not incorporate precursor thymidine but were extremely active in protein biosynthesis. In mechanically released organisms, thymidine incorporation was inhibited immediately by rifamycin (40 microM) and hydroxyurea (10 mM), but it was not affected by chloramphenicol (310 microM) until 4 h after addition of the drug. Incorporation of H3 32PO4 by both populations of organisms was also inhibited by rifamycin, chloramphenicol and hydroxyurea, but the time sequence of inhibition differed. Southern hybridization utilizing 32P-labelled DNA suggested that both populations synthesized authentic chromosomal DNA sequences, as well as QpH1 plasmid DNA, during acid activation of metabolism.     

Cytidine monophosphate-dependent synthesis of phosphatidylglycerol in permeabilized type II pneumonocytes.

Results of previous investigations support the proposition that, in type II pneumonocytes, CMP is involved in integration of the synthesis of phosphatidylcholine and phosphatidylglycerol for lung surfactant. In the present investigation, the amount of CMP in rat type II pneumonocytes was altered directly and resultant changes in the synthesis of phosphatidylglycerol were examined. Type II pneumonocytes were made permeable to CMP by treatment with Ca2+-free medium, and phosphatidylglycerol synthesis was then assessed by measurement of the incorporation of a radiolabelled precursor, [14C]glycerol 3-phosphate, that was not effectively utilized by cells that resisted permeabilization. Incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol (but not into other lipids) was stimulated greatly by CMP (half-maximal stimulation at approx. 0.1 mM). CMP stimulated the incorporation of [14C]glycerol 3-phosphate into both the phosphatidyl moiety and the head group of phosphatidylglycerol. Incorporation of [14C]palmitate into phosphatidylglycerol was also stimulated by CMP. myo-Inositol, at concentrations found in foetal-rat serum (0.2-2.0 mM), inhibited CMP-dependent incorporation of [14C]glycerol 3-phosphate into phosphatidylglycerol and promoted, instead, CMP-dependent incorporation into phosphatidylinositol. These data, when extrapolated to foetal type II pneumonocytes, are consistent with the view that the developmental increase in the synthesis of phosphatidylglycerol for surfactant by foetal lungs is promoted by the increase in intracellular CMP and the declining availability of myo-inositol that were found previously to be associated with this period of development.     

Effect of adrenaline on 32P incorporation into rat fat-cell phospholipids

1. The phospholipid composition of fat-cells prepared from rat epididymal fat-pad was determined. 2. The incorporation of [³²P]P_i into the phospholipids of fat-cells incubated in glucose-free medium and the effect of adrenaline and of α- and β-adrenergic blocking agents, were studied. 3. Incorporation of [³²P]P_i into fat-cell phospholipid increased with time; incubation with adrenaline resulted in increased incorporation that was related to the concentration of adrenaline. 4. The pattern of incorporation of [³²P]P_i into the individual phospholipids of fat-cells after incubation for 1h was determined; adrenaline (5.4μm) resulted in increased incorporation into phosphatidylcholine. 5. Incubation of fat-cells with propranolol (34μm) and adrenaline (5.4μm) resulted in abolition of adrenaline-stimulated lipolysis; there was a decrease in the specific radioactivity of phosphatidylcholine and an increase in the specific radioactivity of phosphatidylethanolamine, phosphatidic acid, phosphatidylinositol and cardiolipin compared with cells incubated with adrenaline alone. 6. Incubation of fat-cells with phenoxybenzamine (0.1mm) and adrenaline (5.4μm) resulted in stimulation of lipolysis, and in diminished specific radioactivities of phosphatidylcholine, phosphatidic acid, phosphatidylinositol, phosphatidylglycerol and choline plasmalogen compared with cells stimulated with adrenaline alone.     

Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of ¹⁴C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of ¹⁴C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone.     

Thymidine metabolism and DNA synthesis in Newcastle disease virus-infected cells.

The inhibition of thymidine incorporation into DNA in Newcastle disease virus-infected cells has been studied. At 6 h after infection of L-929 cells at high multiplicity, transport of exogenous thymidine across the cell membrane was inhibited. The kinetics of this inhibition, decreased Vmax with no change in Km, suggest that there are fewer sites available for transport in infected cells. The conversion of thymidine to dTTP was not inhibited. Equilibrium of exogenous thymidine with the acid-soluble pool occurred more slowly and at a lower level of radioactivity than in uninfected cells, and there was a reduction in the rate of incorporation of exogenous thymidine into DNA. The reduction of incorporation into the pool and into DNA was proportionate. The size of total cellular dTTP pools was changed very little in infected cells. DNA synthesized in infected cells in the presence of [3H]BrdUrd had reduced incorporation of tritium but similar buoyant density to that from uninfected cells. The results show that Newcastle disease virus inhibits DNA synthesis directly and, in addition, decreases thymidine transport. Together these account for the overall decrease in thymidine incorporation into DNA of infected cells.     

In Vitro Polyoma DNA Synthesis: Characterization of a System from Infected 3T3 Cells

A lysate from hypotonically swollen polyoma-infected BALB/3T3 cells incorporated labeled deoxynucleotide triphosphates into both viral and cellular DNAs. The incorporation was stimulated by the presence of ATP, deoxynucleotide triphosphates, thiols, and magnesium ions. Strong inhibition of incorporation was observed with thiol reagents and arabinosyl nucleotide triphosphates. The rate of in vitro synthesis increased with the temperature of incubation as expected. Incorporation into cellular DNA for up to 2 h was observed in lysates from virus-infected and serum-stimulated cells but not from resting cells. Synthesis in the system, therefore, appeared to reflect the physiological state of the cells before preparation of the lysate. Incorporation into viral DNA stopped far sooner than that into cellular DNA. During the initial phase of the in vitro incubation, incorporation occurred into viral replicative intermediates (RI). These RIs had identical properties to those isolated after in vivo pulse labeling and a substantial proportion of them was matured to form I DNA at later times in the incubation through all the stages known to occur in vivo. Density labeling of the in vitro product showed that practically all of the RIs pre-existing in the infected cell took part in the in vitro reaction. Analysis of DNA labeled in vitro in the presence of 5-bromodeoxyuridine triphosphate showed that synthesis occurred on RIs at all stages of replication and that the progeny strands were elongated by up to 80% of unit viral DNA length. Pre-existing RIs, pulse labeled in vivo, showed evidence of a pool at a late stage of replication which required elongation of their progeny strands by approximately 25% during conversion to form I molecules. From density-labeling experiments, we were also able to show that viral DNA synthesis in vitro was semiconservative. The major reason for cessation of viral DNA synthesis in vitro was the very limited ability of the lysate to initiate new rounds of viral DNA synthesis.     

Inhibition of carbohydrate incorporation in transformed cells by a cancer-associated galactosyltransferase acceptor (CAGA)

The effect of cancer-associated galactosyltransferase acceptor (CAGA) on incorporation of a variety of macromolecular precursors has been studied in transformed and nontransformed cells. Incorporation of [3H]-mannose, [3H]-galactose, and [3H]-glucosamine into acid precipitable material after one-hour pulse was inhibited more than 70% within four hours after exposure to CAGA in polyoma-transformed BHK cells and within eight hours after exposure in chick embryo fibroblasts infected with a temperature-sensitive RSV mutant (Ts68) grown at the permissive temperature (CEF-RSV 37 degrees C). Initial short-term rate of uptake (less than one minute) and total long-term uptake (one hour) of the labelled carbohydrates (acid-soluble and acid-insoluble material) was inhibited less than 15% over this period. Incorporation of 14C-leucine, 3H-serine, 3H-uridine, and 3H-thymidine into acid-precipitable material was also inhibited greater than 85% in transformed cells, but more than 12-hour exposure to CAGA was required before maximal inhibition was detected. Uptake of these labelled precursors was inhibited less than 20% up to eight hours after exposure to CAGA. In nontransformed cells (BHK and CEF) incorporation of labelled monosaccharides as well as protein and nucleic acid precursors into acid-precipitable material was reduced less than 25% up to 12 hours following exposure to CAGA. Infected CEF grown at the nonpermissive temperature (CEF-RSV 41 degrees C) were affected to an extent similar to other nontransformed cells. These data suggest that the specific action of CAGA on transformed cells may be due to inhibition of glycoconjugate synthesis.     

Regulation of lipogenesis in mitogen-stimulated human lymphocytes by thyroid hormones

The hormonal regulation of precursor incorporation into cellular lipids has been investigated in human lymphocytes stimulated with phytohemeagglutinine. Addition of thyroxine (5 micrograms/ml) for 72 h increased incorporation of [14C]acetate into the triacylglycerol fraction to 290% above the hormone-free control values. Incorporation into the cholesterol fraction was elevated up to 188% under the same conditions. Triiodothyronine was less effective than thyroxine: maximal effects were 153% of the control for triacylglycerols and 142% for cholesterol. Similar results were obtained when [14C]palmitic acid was used as a precursor for triacylglycerol synthesis. Effects of insulin on the parameters described were less pronounced than those obtained with thyroid hormones. Cellular triacylglycerol and protein contents were not elevated significantly by thyroid hormone addition. Further, incorporation of labelled thymidine, uridine, and leucine into acid-precipitable products was not elevated by triiodothyronine above mitogen-stimulated levels. It is concluded, that rapidly dividing lymphocytes provide a suitable system for studies concerning human lipid metabolism.     

Inhibition of phosphorylation of cellular dUTP nucleotidohydrolase as a consequence of herpes simplex virus infection

During an infection with herpes simplex virus, activity of cellular dUTPase decreases as a function of time, post-infection, while virus-encoded dUTPase activity increases. Prelabeling of cells with 35S-methionine and immunoprecipitation analysis, using monoclonal antibodies, indicates that cellular dUTPase protein levels remain the same (with respect to levels in uninfected cells) throughout the infection period. New synthesis of cellular dUTPase does not occur in infected cells as determined by 35S-methionine labeling during infection. Further characterization of the cellular dUTPase, in uninfected cells, reveals that the protein is post-translationally phosphorylated at serine residues. Pulse labeling of virus-infected cells with 32P-orthophosphate reveals that the phosphorylation rate of the cellular dUTPase protein decreases significantly as a function of time post-infection. In an effort to establish that phosphate turnover was occurring on the cellular dUTPase protein, cells were prelabeled with 32P-orthophosphate and then infected with HSV in the absence of label. Evidence from this experiment indicates that the phosphate moiety is removed from the cellular dUTPase protein during the infection. A series of viable virus mutants was generated by insertional inactivation of the HSV dUTPase gene. These mutants do not express viral dUTPase activity and HSV dUTPase protein is not detected by western blot analysis. However, in contrast to the wild-type situation, these mutant virus retain significant cellular dUTPase activity throughout infection. Interestingly, phosphorylation of cellular dUTPase protein is now readily detectable in each of the mutant virus-infected cells. These studies indicate that cellular dUTPase activity is diminished in wild-type HSV-infected cells by a process of dephosphorylation. It also appears that in mutant HSV, lacking the virus dUTPase, the mechanism of dephosphorylation and thus inactivation of cellular dUTPase is not functional. The end result is that the mutant virus can now rely on the cellular activity for its survival.