An ultramicrochemical test for mycoplasmal contamination of cultured cells.

A sensitive ultramicrochemical enzyme test for mycoplasmal contamination of cultured cells, based on the determination of the activity of adenosine phosphorylase, is described. The test was performed by assaying the enzymatic conversion of [8-14C]adenine and ribose-1-phosphate to [8-14C]adenosine by incubating a plastic leaflet carrying a counted number of cells (1 to 10). These leaflets were isolated from the bottom of the same plastic film dish in which the cells were cultured for experimental or diagnostic purposes, e.g. prenatal diagnosis or inborn errors of metabolism. The present test should be several 1000-fold more sensitive than the originally reported enzymatic method because (a) the adenosine-phosphorylase reaction is measured in the nucleoside forming direction which is by far the most active; and (b) the assay is performed with the cells and not with the culture medium. The latter is of special importance for the detection of those low-grade contamination in which most of the mycoplasma particles are attached to cell membranes.     

Comparison of adenosine metabolism in leaves of several mangrove plants and a poplar species

A possible increased demand for ATP in salt- tolerant mangrove plants was studied by the comparison of metabolic fates of [8-¹⁴C] adenosine in leaf disks of several mangrove plants and of poplar. In mangrove trees, Rhizophora stylosa, Bruguiera gymnorrhiza, Kandelia candel and Sonneratia alba, 56–92% of [8-¹⁴C]adenosine taken up by leaf disks was converted during 3 h incubation to salvage products, i.e., nucleotides and RNA. Synthesis of nucleotides including ATP was stimulated by salt stress induced by 250 mM NaCl. In leaf disks of Avicennia marina, a mangrove shrub that produces glycinebetaine as compatible solutes, 46% of radioactivity entered salvage products when [8-¹⁴C] adenosine was continuously supplied to the leaf disks. Hydrolysis of adenosine to adenine was extremely active in this mangrove shrub. This is probably due to the high activity of adenosine nucleosidase (EC 3.2.2.7). In leaf disks of another mangrove shrub, Lumnitzera racemosa, only limited amounts of [8-¹⁴C]adenosine were metabolised (< ca. 30% taken up by leaf disks), but synthesis of ATP and ADP was stimulated by salt stress. In Pemphis acidula leaf disks, adenosine salvage activity was low and more than 30% of adenosine was hydrolysed to adenine. In leaf disks of poplar, a non-salt-resistant plant, ca. 40% of [8-¹⁴C] adenosine was converted to salvage products during 3 h of incubation, but the rate was slightly reduced by treatment with 250 mM NaCl. The present results suggest that large mangrove trees generally have efficient adenosine salvage ability, which is stimulated by salt. Lesser salvage activity is found in small size mangrove shrubs, although salt generally still enhances salvage activity.     

Stimulatory effect of gonadotropins on the synthesis of adenosine 3′ :5′-cyclic monophosphate and progesterone by suspensions of rat ovarian interstitial cells

A cell suspension was prepared from immature rat ovaries by treatment with trypsin and collagenase. The isolated cells were capable of converting [8-¹⁴C]adenine to cyclic [¹⁴C]AMP and the rate of this conversion was stimulated in vitro by luteinizing hormone and human chorionic gonadotropine, but not by prolactin, norepinephrine, dopamine or albumin. The accumulation of progesterone was also measured in these cells by radioimmunoassay. In vitro addition of luteinizing hormone stimulated the accumulation of radioimmunoassayable progesterone. The conversion of [8-¹⁴C]adenine to cyclic [¹⁴C]AMP showed a rapid increase during the first 30 min of the incubation period when luteinizing hormone was added to the incubation medium. Progesterone accumulation in response to the same dose of luteinizing hormone showed a lag period for the first 30 min of incubation after which there was an increase up to 2 h. The luteinizing hormone-induced progesterone accumulation was sensitive to puromycin, but there was no effect on the luteinizing hormone-induced increase in cyclic [¹⁴C]AMP formation from [8-¹⁴C]-adenine. Actinomycin D also inhibited the luteinizing hormone-induced progesterone accumulation, with no effect on cyclic AMP formation. The results suggest that the luteinizing hormone-induced progesterone accumulation in rat ovarian interstitial cell suspension is preceded by an increased accumulation of cyclic AMP and that the accumulation of steroid under the influence of luteinizing hormone involve processes sensitive to puromycin and antinomycin D.     

A sensitive fluorimetric procedure for the determination of aliphatic epoxides under physiological conditions

Ribose 1-phosphate has been measured in rat tissues by an enzymatic radioactive assay. The sugar phosphate is converted into [¹⁴C]inosine via the two following combined reactions: ribose 1-phosphate + [¹⁴C]adenine ? [¹⁴C]adenosine + phosphate (adenosine phosphorylase); [¹⁴C]adenosine + H₂O → [¹⁴C]inosine + NH₃ (adenosine deaminase). Tissue extracts are incubated in the presence of excess [¹⁴C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of ribose 1-phosphate present in tissue extracts. Liver was found to contain the highest level of ribose 1-phosphate (ca. 800 nmol/g wet wt).     

A rapid and simple radioassay for inosinic acid: pyrophosphate phosphoribosyltransferase in the presence of xanthine oxidase and vice versa.

A simple and rapid technique for measuring IMP:pyrophosphate phosphoribosyltransferase (HPRibTase) activity of rat intestinal homogenates, in the presence of xanthine oxidase, is described. By introducing 2.5 × 10^?5m allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine) into the reaction mixture, the [8-¹⁴C]hypoxanthine (Hx) is converted only to [8-¹⁴C]inosinic acid (IMP). The xanthine oxidase activity is completely inhibited under this condition. When xanthine oxidase is not blocked, diversion of substrate to urate can invalidate assays of HPRibTase.Using [8-¹⁴C]Hx as substrate, in the presence and absence of allopurinol, the activity of both HPRibTase and xanthine oxidase of the same tissue homogenate is determined. We have simplified the conventional chromatographic separation of the reactant products by spotting the reactant on DEAE cellulose paper followed by repeated washings with 4 mm ammonium formate solution. The unreacted radiosubstrate is washed off, and the [8-¹⁴C]IMP or [8-¹⁴C]uric acid formed remains adsorbed on the paper. The major advantages of this method are speed, reproducibility, sensitivity, ability to process many samples, and a low blank value.Our studies on the enzyme distribution along the intestinal villus have shown that while most of the HPRibTase activity is associated with rapidly multiplying crypt cells, the xanthine oxidase activity is more evenly distributed along the villus, and the activity is effected more by exongeneous effectors. The colon has the highest HPRibTase and lowest xanthine oxidase activity of all the intestinal mucosa cells. Small bowel mucosa is high both in xanthine oxidase and HPRibTase.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin fibroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

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.     

Experimental modulation of PRPP availability for ribonucleotide synthesis from hypoxanthine in human skin febroblast cultures

The intracellular concentration of the cosubstrate 5-phosphoribosyl 1-pyrophosphate (PRPP) may be rate-limiting for the reactions, catalysed by hypoxanthine phosphoribosyltransferase, by which mammalian cells convert the purine bases hypoxanthine, xanthine, and guanine to their ribonucleotide derivatives. The rate of conversion of [¹⁴C]hypoxanthine to radioactive phosphorylated products by intact human diploid skin fibroblasts was measured in the presence of compounds previously reported to alter PRPP concentration in a variety of cell types Methylene blue, previously reported to increase PRPP concentration in a variety of cultured cells including skin fibroblasts, increased product formation from hypoxanthine, with maximum effect following 60 min preincubation with 0.4 mM. Incubation with adenine, orotic acid, allopurinol, or adenosine has been shown to decrease PRPP concentration. Of these compounds, only adenine and adenosine decreased the rate of ribonucleotide synthesis from hypoxanthine in cultured skin fibroblasts. This decrease probably resulted from decreased PRPP synthesis rather than increased PRPP utilization. The reaction products isolated from cells following incubation with either [¹⁴C]adenine or [¹⁴C]adenosine included adenosine monophosphate and adenosine diphosphate, both inhibitors of PRPP synthetase.     

Radioactive measurement of 2' ,3'-cyclic nucleotide 3'-phosphodiesterase activity in the central and peripheral nervous system and in extraneural tissue.

This paper describes a new, sensitive, and reproducible method for the determination of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (EC 3.1.4.37) activity in both central and peripheral nervous system tissue, as well as in extraneural tissue. Radioactive [8-³H]adenosine 2′,3′-cyclic monophosphate was used as the substrate. The [8-³H]2′-AMP product formed was isolated by thin-layer chromatography, or, alternatively, the reaction was coupled with an excess of Escherichia coli alkaline phosphatase, and the [8-³H]adenosine formed was isolated by column chromatography on AG 1-X2 resin. The values obtained by the two methods were compared with those obtained using a spectrophotometric method. Hydrolysis rates of 0.50 nmol/min could be reproducibly measured in 18-day fetal rat spinal cord.     

trans-Ribosylzeatin: Its Biosynthesis in Zea mays Endosperm and the Mycorrhizal Fungus, Rhizopogon roseolus

When [8-¹⁴C]-N⁶-(Δ²-isopentenyl) adenosine is incubated with the endosperm of corn (2 weeks after pollination), it is converted to [¹⁴C]-N⁶-(4-hydroxy-3-methylbut-2-trans-enyl) adenosine, trans-ribosylzeatin. This biosynthetic step, N⁶-(Δ²-isopentenyl) adenosine to ribosylzeatin, also occurs in the mycorrhizal fungus, Rhizopogon roseolus.     

A batch procedure for the assay of cyclic nucleotide phosphodiesterase.

A procedure for the assay of cyclic nucleotide phosphodiesterase is described in which labeled cyclic nucleotide is separated from labeled nucleoside by the batchwise addition of ethanolic slurries of Dowex 2 fluoride. Under the conditions described there is no detectable adsorption of nucleoside by the anion exchanger, which removes more than 95% of the tritium in boiled samples of [8-³H]cAMP or [8-³H]cGMP. Linear time courses and enzyme vs activity relationships are described for 10^?3 and 10^?7m cAMP and 10^?4m cGMP. The method is limited by interference by neutral salts and by the enzymatic conversion of adenosine into inosine.     

Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS

Uptake and degradation of the cytokinin, N⁶-(Δ²-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N⁶(Δ²-isopentenyl)[8-¹⁴C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N⁶-(Δ²-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-¹⁴C]-N-⁶(Δ²-isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N⁶(Δ² isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N⁶-(Δ²-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin.     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Excreted adenosine is a cell density signal for the initiation of fruiting body formation in Myxococcus xanthus

Myxococcus xanthus is a bacterium that forms multicellular fruiting bodies in response to starvation. The initiation of fruiting body formation is cell density dependent, and we suggest that cells measure their cell density by titering the extracellular concentration of excreted adenosine. Our evidence is as follows: (1) At low cell densities fruiting body formation does not occur unless adenosine is added. (2) Norit, a substance that binds purines, inhibits fruiting body formation, and this inhibition is reversed by adenosine. (3) Cells labeled with [¹⁴C]carbonate excrete [¹⁴C]adenosine which is bound by the Norit. Furthermore, [¹⁴C]adenosine is excreted by developing cells at a concentration that will induce fruiting body formation at low cell density. The extracellular adenosine concentration increases with the cell density over a broad range of densities. (4) Hadacidin, an inhibitor of de novo AMP synthesis, inhibits fruiting body formation, and inhibition by hadacidin can be reversed with adenosine. Adenosine also appears to be involved in the aggregation process because the shape and size of the fruiting bodies are sensitive to the external concentration of adenosine.     

Primary cultures of heart cells from the scallp Pecten maximus (mollusca-bivalvia)

Summary Primary cultures of Pecten maximus heart cells, isolated by an enzymatic procedure, were routinely obtained with a high level of reproducibility in a simple medium based on sterile seawater. Cells attached to the plastic substratum without the need to add a special factor. The number of adhering cells gradually increased with the time of culture. Two types of adhering cells were observed: epitheliallike cells and fibroblastlike cells, which were more numerous. The latter cells were identified as myocytes by electron microscopy and immunofluorescent staining. Results obtained by autoradiography, after incorporation of [¹⁴C]leucine, [³H]thymidine, and [¹⁴C]acetate, confirmed functional activity of the cells. These cultures were maintained viable in vitro during at least 1 mo.     

Conversion of diglyceride to triglyceride by rat liver microsomes: a requirement for the 105,000 x g supernatant

This paper describes a requirement for the 105,000 × g supernatant of rat liver for the synthesis of triglyceride from diglyceride and palmityl coenzyme A by rat liver microsomes. ATP and magnesium chloride are also required. The incorporation of both [1-¹⁴C]-palmityl coenzyme A and [1-¹⁴C]-diolein into triglyceride has been observed. The 105,000 × g supernatant has no enzymatic activity for this reaction when incubated in the absence of microsomes. The supernatant contains a soluble, essential protein which is nondialyzable, heat sensitive, and destroyed by trypsin. Net synthesis of triglyceride has been demonstrated by chemical analysis.     

Caffeine biosynthesis in Camellia sinensis

The metabolism of adenine and guanine, relating to the biosynthesis of caffeine, in excised shoot tips of tea was studied with micromolar amounts of adenine-[8-¹⁴C] or guanine-[8-¹⁴C]. Among the presumed precursors of caffeine biosynthesis, adenine was the most effective, whereas guanine was the least effective. After administration of a ‘pulse’ of adenine-[8-¹⁴C], almost all of the adenine-[¹⁴C] supplied disappeared by 30 hr, and ¹⁴C-labelled caffeine and RNA purine nucleotide (AMP and GMP) synthesis increased throughout the experimental period, whereas the radioactivities of free purine nucleotides, 7-methylxanthine and theobromine increased during the first 10 hr incubation period, followed by a steady decrease. By contrast, more than 45% of the guanine-[8-¹⁴C] supplied remained unchanged even after a 120 hr period. The main products of guanine-[8-¹⁴C] metabolism in tea shoot tips were guanine nucleotides, theobromine, caffeine and the GMP of RNA. The results support the hypothesis that the purine nucleotides are synthesized from adenine and guanine via the pathway of purine salvage. Adenylate is readily converted into other purine nucleotides, whereas the conversion rate of guanylate into other purine nucleotides is very low.The results also support the view that 7-methylxanthine and theobromine are precursors of caffeine. For the origin of the purine ring in caffeine, purine nucleotides in the nucleotide pool rather than in nucleic acids are suggested.     

Increased arginase activity during lymphocyte mitogenesis

A sensitive assay for arginase activity was developed using [guanidino-¹⁴C]arginine as substrate and measuring the production of ¹⁴CO₂ from [¹⁴C]urea in the presence of urease. Arginase activity was measured in bovine lymphocytes after activation by Concanavalin A. The specific enzymatic activity of arginase doubled in 6 hours and increased nearly 4-fold by 24 hours after stimulation. It is suggested that the role of arginase in these cells is to provide ornithine as substrate for the synthesis of putrescine, precursor of the polyamines spermidine and spermine.     

Enzymatic isomerization (Δ7 → Δ8) of the nuclear double bond of 14α-alkyl substituted sterol precursord of cholesterol

The catalysis by rat liver microsomes under anaerobic conditions, of the conversion of [3α-³H]14α-methyl-5α-cholest-7-en-3β-ol and of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol to labeled 14α-methyl-5α-cholest-8-en-3β-ol and 14α-hydroxymethyl-5α-cholest-8-en-3β-ol, respectively, has been demonstrated. This finding is of importance in evaluating past research in this area and in consideration of pathways and mechanisms involved in enzymatic removal of carbon atom 32 of 14α-methyl sterols. Also described herein are syntheses of [2,4-³H]14α-hydroxymethyl-5α-cholest-7-en-3β-ol and 3β-acetoxy-14α-methyl-5α-cholest-8-ene.     

Effects of benzimidazole on the purine and pyrimidine metabolism of yeast

Yeast cells inhibited by benzimidazole accumulate hypoxanthine with an associated efflux of xanthine. Unlike control cells, inhibited cells contain no detectable free UMP and CMP. Benzimidazole decreases uptake of [8-¹⁴C]-hypoxanthine into the intracellular pool of hypoxanthine and xanthine but causes radioactive xanthine to accumulate in the medium. In inhibited cultures there is a threefold increase in incorporation of [8-¹⁴C]hypoxanthine into the total (intracellular plus extracellular) xanthine. Uptake of [8-¹⁴C]hypoxanthine into free nucleotides and into bound adenine and guanine was inhibited by 70%. Uptake of [U-¹⁴C]glycine into IMP, AMP, GMP, DNA and RNA was also substantially decreased. Incorporation of [2-¹⁴C]uracil into the intracellular uracil pool was inhibited by 30% and into free uridine and cytidine by over 90%. Benzimidazole inhibited incorporation of [8-³H]IMP into AMP and GMP, and decreased substantially the activity of glutamine-amidophosphoribosyltransferase (EC 2.4.2.14). Yeast cultures were shown to N-ribotylate benzimidazole. Results are consistent with benzimidazole inhibiting yeast growth by competing for P-rib-PP and so depriving other ribotylation processes such as the ‘salvage’ pathways and de novo synthesis of purines and pyrimidines.