Radiolabelled Precursors Research Articles

The contribution of host and pathogen to ethylene biosynthesis in Penicillium digitatum-infected citrus fruit

The biosynthesis of ethylene by grapefruit (Citrus paradisi Macf) cv. “Marsh Seedless” 6 days after inoculation with either an ethylene producing or a non-ethylene-producing isolate of Penicillium digitatum, was studied. Relatively low levels of ethylene were produced by the apparently healthy region, at the edge of rot in fruits infected with either the ethylene producing or the nonethylene-producing isolate of the fungus. Higher levels of production were found in regions of the peel showing infection symptoms, but only in fruit infected with the ethylene-producing isolate. The production of symptoms and the ACC content of the peel of fruits inoculated with the ethylene-producing isolate was similar to that in fruits inoculated with the non-producing isolate.The use of radiolabelled precursors showed that the ethylene produced by the healthy parts of the fruit originated from methionine, while the ethylene produced by the infected parts of the fruit originated mostly from glutamic acid. Furthermore, ethylene production by the healthy part of the fruit was markedly enhanced by 1-aminocyclopropane-1-carboxylic acid (ACC), and to a lesser extent by CuSO4, but it was inhibited by aminoethoxyvinylglycine (AVG). In contrast the production of ethylene by infected peel and by P. digitatum in culture was not affected by ACC, but was markedly inhibited by CuSO4, and, to a lesser extent, by AVG.These results suggest that ethylene production in the healthy part of the fruit is of plant origin, whereas the markedly enhanced production of ethylene by the P. digitatum-infected regions, is mostly or entirely offungal origin.

Conversion of a radiolabelled ecdysone precursor, 2,22,25-trideoxyecdysone, by embryonic and larval tissues of Locusta migratoria

A high specific activity tritiated ecdysone precursor, 2,22,25-trideoxyecdysone, was used to probe the capacity of various embryonic and larval tissues to perform the last 3 hydroxylation steps in ecdysone biosynthesis. Embryos at early stages of development, prior to the differentiation of their endocrine glands and embryonic heads, thoraces and abdomens of later stages, were found to have the capacity to hydroxylate the precursor to ecdysone. Larval epidermis and fat body are also able to transform 2,22,25-trideoxyecdysone into ecdysone; Malpighian tubules and midgut hydroxylate the precursor at C-2 but are apparently unable to hydroxylate both at C-22 and C-25. Larval prothoracic glands convert the precursor to ecdysone at a very efficient rate, which is 1–2 magnitudes higher than that of the other tissues investigated; several data argue for the existence of a privileged sequence of hydroxylations, C-25, C-22, C-2, in the larval prothoracic glands.

1469 EFFECT OF RITODRINE ON BILIRUBIN FORMATION IN THE NEONATAL RAT

Ritodrine hydrochloride (RH) is a β-adrenergic, tocolytic agent. RH activates adenyl cyclase which generates cyclic adenosine monophosphate (cAMP). cAMP is reported to increase hepatic heme oxygenase (HO) activity. Because increased hepatic HO activity has been associated with increased hepatic early-labeled bilirubin (ELB) production, we studied the effect of RH on total bilirubin formation (TBF). Litters of Wistar rats (< 12 hrs old) were divided into groups (3 rats each) of equal weight and given 2 SQ injections of RH (35 mg/kg) or saline at 7 and 5 hrs prior to injection of a radio-labeled hepatic heme precursor, δ-ALA-5-14C (0.6 μCi/12.3 nmol) (t=0). 14CO was collected continuously and was quantitated at t=1, 2, 3, 4, 6, 8, and 12 hrs. The excretion rate of carbon monoxide (VeCO), an index of TBF, was determined at t=0, 2, 4, 6, 8, and 12 hrs. At t=12 hrs, the animals were decapitated and blood and liver were collected. Plasma bilirubin (mg/dl) was measured by a UB Analyzer and HO activity(nmoles bili/min/10 mg prot) was determined spectrophotometrically. Our results suggest that, although high dose RH administration in rats increases hepatic HO activity and ELB, these changes are minimal and the effect of RH on TBF is small.

Intracellular activity of HPRT Cape Town: purine uptake and growth of cultured cells in selective media.

The low activity of the human variant HPRT Cape Town is associated with substrate inhibition by hypoxanthine and guanine in vitro. The intracellular activity of this variant was investigated by study the relative uptake of radiolabelled purine nucleotide precursors and the growth in selective media of EBvirus-transformed lymphoblasts prepared from the proband (TK). These cells incorporated less than 10% of the [14C]hypoxanthine and [14C]guanine of the control cells; while their purine de novo synthesis was accelerated 8-fold. In selective media the HPRT Cape Town cells grew in a similar manner to HPRT-ve cells. These results indicate that if substrate inhibition is responsible for the low intracellular activity of HPRT Cape Town, the concentration of either hypoxanthine or guanine in the vicinity of the active site of the enzyme must be greater than the Ki(app) for these substrates, 118 and 28 mumol L(-1) respectively. Evidence is presented that the intracellular concentration of guanine, but not hypoxanthine, is well in excess of the Ki(app) in cultured lymphoblasts.

Neutral glycosphingolipids of Fabry's disease lymphoblastoid lines established by Epstein-Barr virus transformation

Human lymphoid cell lines established by Epstein-Barr viral transformation of peripheral B-lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors. Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose. Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the 'pulse') was studied after cultivating the cells without radiolabelled precursors ('chase'). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half-life time around 15-25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of alpha-galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445-456] and demonstrates that alpha-galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).

Synthesis of prostaglandin E2 and F2 alpha in human tonsillar lymphocytes.

Although prostaglandins (PGs), especially PGE2 and PGF2 alpha, are thought to be associated with inflammation, there have been no papers dealing with the synthesis of PGE2 and PGF2 alpha in human tonsillar lymphocytes. The features of PG synthesis in lymphocytes were investigated by incubating cells with radiolabelled precursors. Synthesis of PGE2 was greater than that of PGF2 alpha in tonsillar lymphocytes. PGE2 was the predominant product in the tonsillar lymphocytes of the recent infection group. There was no significant difference between T and B lymphocytes in the ratio of PGE2 and PGF2 alpha formed. A significant increase in PGE2 formed with bradykinin was found in the tonsillar hypertrophy group. The possible role of PGs in the pathogenesis and mechanism of releasing free fatty acid PG precursors from cell membranes was discussed.

Differential responses of a range of photosynthetic tissues to a substituted pyridazinone, sandoz 9785. Specific effects on fatty acid desaturation

The effect of a substituted pyridazinone (4-chloro-5(dimethylamino)-2-phenyl-3(2H)pyridazinone; Sandoz 9785; BASF 13-338) on the formation of fatty acids from radiolabelled precursors has been studied in a number of angiosperms, bryophytes and algae. The labelling of [ 14C]linolenic acid was decreased by the herbicide in leaves of barley and rye grass and in cucumber cotyledons regardless of whether [ 14C]acetate,[ 14C]oleate or [ 14C]linoleate was used as precursor. A commensurate increase in the labelling of [ 14C]linoleic acid was also observed in these species. In contrast, the pattern of fatty acid labelling in maize, pea and spinach leaves was unaffected by 0.1 mM Sandoz 9785. More generalized inhibition of the incorporation of radioactivity from [ 14C]acetate into the fatty acids of bryophytes and algae was seen. Sandoz 9785 did not alter the distribution of radioactivity in different lipid classes of higher plant leaves, nor did it change the proportions of radioactive fatty ac ids in phosphatidylcholine. In contrast to phosphatidylcholine, which never contained more than trace amounts of [ 14C]linolenate, diacylgalactosylglycerol contained high levels of the radioactive acid. The relative labelling of linolenate was severely reduced in diacylgalactosylglycerol by Sandoz 9785 in sensitive angiosperms. Uptake studies, in which [ 3H]Sandoz 9785 was employed demonstrated that the uptake of Sandoz 9785 was reflection of water uptake. Following its uptake, Sandoz 9785 was rapidly converted into other compounds in pea but only gradually metabolized in cucumber and ryegrass. The results are interpreted as showing, firstly, that the different sensitivity of higher plants to Sandoz 9785 is due to variations both in uptake and in metabolism. Secondly, Sandoz 9785 specifically inhibits the desaturation of linoleate to linolenate and, thirdly, diacylgalactosylglycerol plays a role in this conversion.

Effect of sulfolipid antibodies on the macromolecular synthesis of Mycobacterium smegmatis ATCC607

The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like 3H-thymidine 14C-uracil, 14C-leucine and 14C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.

Polyanions in syphilis: evidence that glycoproteins and macromolecules resembling glycosaminoglycans are synthesised by host tissues in response to infection with Treponema pallidum.

We investigated by means of radiolabelled precursors the source and nature of the polyanionic macromolecules present in rabbit tissues during active syphilis infection. Previous studies indicated that Treponema pallidum itself does not synthesise glycosaminoglycans, at least in vitro. In replicate experiments on unilaterally infected rabbits, tissue from the orchitic testis incorporated two to three times more 35S-sulphate and 3H-glucosamine (on a wet weight basis) than tissue from the non-orchitic contralateral testis. Incorporation of 35S-sulphate was independent of the number of viable T pallidum organisms present in the infested tissue, which suggested that incorporation represented biosynthesis by the host and not the treponeme. Testes from syphilitic rabbits two days after treatment with high doses (100 mg/kg) of penicillin incorporated less 35S-sulphate than untreated infected testes, but more than normal uninfected rabbit testes. This suggests that active syphilitic infection was necessary for maximum biosynthesis of the macromolecule(s) by host tissue. Hydrodynamic profiles showed incorporation of radiolabelled precursors into two distinct fractions of different sizes, which may represent a proteoglycan and a sulphated glycoprotein. Alcian blue staining of syphilitic testes at or after peak orchitis showed focal deposition of newly synthesised polyanionic components during peak orchitis and a more generalised fibrosis in testes after peak orchitis.

Effect of sulfolipid antibodies on the macromolecular synthesis ofMycobacterium smegmatisATCC607

The biosynthesis of DNA, RNA, proteins and lipids in the presence of antiserum to sulfolipids was investigated by studying the incorporation of radiolabelled precursors like 3H-thymidine 14C-uracil, 14C-leucine and 14C-Acetate into their respective macromolecules. Antiserum to sulfolipids had a inhibitory effect on the biosynthesis of all these components. Antiserum also exhibited a growth inhibitory effects as compared to normal serum.

Treponema pallidum does not synthesise in vitro a capsule containing glycosaminoglycans or proteoglycans.

Treponema pallidum was investigated for its ability to synthesise glycosaminoglycans or proteoglycans in vitro. Isolated viable T pallidum organisms were incubated with radiolabelled precursors of glycosaminoglycans, sodium 35S-sulphate and 3H-glucosamine (tritiated glucosamine). T pallidum failed to incorporate sodium 35S-sulphate but did incorporate 3H-glucosamine into a macromolecule which may be associated with the surface of the treponeme. This macromolecule was resistant to degradation by specific glycosaminoglycanases. We conclude that T pallidum does not synthesise a capsule containing glycosaminoglycans in vitro.

Free sterols and glycolipids in the aleurone tissue of germinating wheat

The action of gibberellic acid on wheat aleurone tissue led to reduced levels of free sterols and glycolipids compared with control tissue. Radio-labelled precursors were not incorporated into sterols or glycolipids, although mevalonate and glycerol were incorporated into polyisoprenoid hydrocarbon and phospholipid respectively. It is concluded that sterols and glycolipids are not synthesized in the tissue during germination; this is in contrast to earlier reports of the active, gibberellin-regulated metabolism of phospholipids.

Biosynthesis and transport of gangliosides in peripheral nerve.

Radiolabelled glucosamine was injected into L-7 dorsal root ganglion (DRG) of rabbits. At several different times after injection DRG, lumbosacral trunks (LST) and sciatic nerves (SN) removed and gangliosides extracted. Two and 3 weeks after injection the amounts of radioactivity in the ganglioside fractions of LST SN were significantly higher than at days 1 and 2. The TCA soluble radioactivity decreased dramatically over the same time period. Colchicine prevented the appearance of radiolabelled lipid in LST and SN. From these experiments we conclude that some ganglioside is synthesized in the neuronal cell bodies of DRG and transported in the axons of the sciatic nerve. In another experiment the sciatic nerve was transected and ends separated to prevent regeneration. Ganglioside synthesis and transport were studied in these animals the same way as the previous experiment. There was no difference the amount of radiolabelled ganglioside that was isolated from DRG or LST of transected compared with control nerves. The behavior of several potential acid soluble contaminants was studied in several steps used to isolate gangliosides. Of those studied only CMP-NeuAc could cause significant contamination of the final ganglioside preparation.

Sympathetic denervation impairs agonist-stimulated phosphatidylinositol metabolism in rat parotid glands.

Substance P, muscarinic and alpha-adrenoceptor agonists stimulated the incorporation of [3H]inositol into phosphatidylinositol in rat parotid gland slices. Surgical denervation of the sympathetic input to the rat parotid gland by superior cervical ganglionectomy produced marked reductions in these responses. The stimulated incorporation of radiolabelled precursors into phosphatidylinositol is a measure of its resynthesis after receptor-mediated breakdown of inositol phospholipids. We therefore examined the enzymic site of the lesion induced by sympathetic denervation using parotid gland slices labelled with either [3H]inositol or [32P]phosphate and stimulated with substance P. Receptor-activated phospholipase C attack upon [3H]inositol phospholipids was assayed by measuring the formation of [3H]inositol 1-phosphate in the presence of 10 mM-Li+ to inhibit further breakdown. It was not affected by denervation. Substance P elicited a rapid breakdown of phosphatidylinositol 4,5-bisphosphate and this response was reduced in the denervated gland. The second step in stimulated phosphatidylinositol turnover, phosphorylation of diacylglycerol to phosphatidate was not affected by denervation. Sympathetic denervation appears to induce a specific enzymic lesion in the parotid gland that impairs receptor-stimulated resynthesis of phosphatidylinositol from phosphatidate. This change in membrane lipid metabolism may be related to a number of the effects of sympathetic denervation, such as agonist supersensitivity, reduced gland cell proliferation and induction of new surface receptors.

The occurrence and synthesis of octopamine and catecholamines in the cestode Hymenolepis diminuta

The occurrence and regional distribution of octopamine and catecholamines in the cestode Hymenolepis diminuta were determined by specific radioenzymatic assays. Octopamine was found in the anterior and middle regions (2630 ± 490 and 520 ± 290 pg/mg protein, respectively) but not in the posterior region. On the other hand, dopamine was detected in the anterior region only (4.65 ± 1.42 pg/mg protein). Adrenalin and noradrenalin were not detected in any of the regions. The ability of intact H. diminuta to synthesize octopamine, dopamine and noradrenalin was investigated by incubating the worms in vitro in radiolabelled precursors, followed by separation of the 3H-labelled products by thin-layer chromatography. Only traces of [ 3H]dopamine and [ 3H]noradrenalin were produced in the tissues following incubation in [ 3H]tyrosine or [ 3H]tyramine. However, substantial amounts of [ 3H]octopamine were synthesized from both radiolabelled tyrosine and tyramine. These findings are discussed in the light of octopamine as a possible neurotransmitter/neuromodulator in H. diminuta.

The distribution of precursors of glycoconjugate synthesis in human epidermis.

Biochemical studies [1, 2] have shown that a complex mixture of epidermal glycoconjugates are synthesized when skin slices are incubated in medium containing radiolabelled precursors. However, the products of the whole epidermis were examined in these biochemical studies and no information was provided on the contribution of the different cell layers to the total glycoconjugates synthesized. There are a number of reports of changes in cell surface glycoconjugates with differentiation in other cell types such as lymphoid cells [3] and intestinal epithelial cells [4]. In the epidermis studies using fluorescein-labelled lectins have revealed differences in the lectin-binding properties of different populations of cells [5, 6]. As part of a larger study on the changes in epidermal glycoconjugate synthesis with differentiation and their importance in cell adhesion we now report on the distribution of radiolabelled precursors incorporated into glycoconjugates in the human epidermis. Thin sheets of skin were taken from the thighs of normal adult subjects, who had given their informed consent, using a Davies dermatome and divided into rectangular fragments (1 x 0.5 cm). The fragments were incubated at 371C in 0.2 ml Eagle's minimum essential growth medium containing 2.8 x 10-4 M ascorbic acid, 5 % (v/v) foetal calf serum and one of the radiolabelled precursors, D-(I-3H) glucosamine (50 gCi/ml), L-(1-3H) fucose (50gCi/ml) or 35SO2(165 gCi/ml). In some experiments tunicamycin (2 gg/ml) was added to the medium. After incubation for 24h the medium was removed and the skin fragments were washed five times

Extensive destruction of newly synthesized casein in mammary explants in organ culture.

1. Explants of mammary glands of mid-pregnant rabbits that had been cultured for 18h in the presence of insulin, prolactin and cortisol were incubated at 37 degrees C for 2h in Medium 199 containing l-[4,5-(3)H]leucine. After a wash procedure at 4 degrees C, explants were re-incubated at 37 degrees C in fresh medium and the radioactivity of casein polypeptides isolated by isoelectric focusing (at pH 4.6) was followed with time. Casein radioactivity rose during the first hour of re-incubation, but fell markedly during the subsequent hour. 2. Loss of radioactivity represented casein degradation, since less than 10% of newly synthesized casein was found in the incubation medium. 3. Such a loss of radioactivity was not due solely to hydrolysis of signal peptides, since similar results were obtained when l-[5-(3)H]proline, which is not part of casein signal peptides, was the radiolabelled precursor. 4. A dual-isotope experiment using l-[U-(14)C]proline and N-[(3)H]acetyl-d-mannosamine gave similar profiles of radioactivity loss from isoelectrically focused casein, indicating that degradation of mature casein was occurring. 5. Analysis of total pellet and particle-free-supernatant fractions prepared by centrifugation of explant homogenates at 115000g(av.) for 1h did not show loss of radioactivity on re-incubation. Total pellet-protein radioactivity remained constant, whereas total soluble-protein radioactivity increased during the 2h re-incubation period. 6. Radioactivity in a specific particle-free-supernatant polypeptide, the subunit of fatty acid synthetase, mimicked that of the total soluble protein. 7. Addition of cycloheximide (20mug/ml) during the re-incubation period completely blocked the incorporation of radioactivity from l-[5-(3)H]proline into casein and the subsequent fall, indicating that observations were being made on newly synthesized casein. 8. Addition of chloroquine (50mum) did not prevent the increase in radioactivity from l-[5-(3)H]proline into casein during the first hour of re-incubation, but did prevent the loss of radioactivity in the second hour. 9. The intracellular degradation of a newly synthesized milk protein is discussed in relation to the known intracellular degradation of other secretory polypeptides.

Fate of the glycerol and acyl moieties from dietary trioleoylglycerol in the sea star, Asterias rubens

1. 1. The metabolic fate of the acyl and glycerol moieties of dietary triacylglycerols was studied in the female sea star Asterias rubens, 1, 6 and 24 hr after oral administration of radiolabelled precursors. 2. 2. In the lipids of the pyloric caeca, ovaries and coelomocytes only low percentages of recovery (<10% of the dose) were found, indicating that much of the precursor remains elsewhere in the digestion system or that the acyl moieties have been used for energy production. 3. 3. Changes occurring in the 3H/ 14C ratio of all lipid fractions suggest a “sparing effect” for fatty acids. 4. 4. Differences were found at various stages of gonadal development and also in the distribution pattern of the radiolabel over individual lipid classes from pyloric caeca, ovaries and coelomocytes. 5. 5. High radioactivities found in coelomocyte lipids may point to a rapid turnover of structural lipid or some role of coelomocytes in lipid transport.

The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks

Irvin A.D., Boarer C.D.H., Kurtti T.J. and Ocama J.G.R. 1981. The incorporation of radio-labelled nucleic acid precursors by Theileria parva in bovine blood and salivary glands of Rhipicephalus appendiculatus ticks. International Journal for Parasitology 11:451–456. The uptake of radio-labelled nucleic acid precursors by blood and tick salivary gland forms of Theileria pana was studied. Piroplasms took up tritiated purines, particularly hypoxanthine, but not pyrimidines. Similar uptake was recorded by T. parva, both in tick saliva and in salivary glands maintained in vitro. Intermediate parasite stages were those most readily labelled in glands; this reflected active nucleic acid synthesis associated with rapid parasite division. Radio-labelling of T. parva in tick salivary glands could be of value in procedures used for concentrating and purifying theilerial sporozoites.

The effect of insulin-like growth factor (IGF) and of human serum on steps in proteoglycan synthesis.

Embryonic chick pelvic cartilages were incubated in the presence of insulin like growth factor (IGF) (1 - 100 microunits/ml), as well as normal human serum (5%), with radiolabelled precursors of proteoglycan (PG) synthesis: L-[3-3H]serine, D-[6-3H]glucosamine and [35S]Na2SO4. IGF alone (1 - 15 microunits/ml), stimulated in a dose-dependent manner D-[6-3H]glucosamine incorporation into tissue-bound and soluble isolated glycosaminoglycan (GAG) chains. L-[3-3H]serine incorporation into PG molecules was not stimulated by IGF (1 - 100 microunits/ml), despite the increase in the uptake of this precursor into intact cartilage. [35S]Na2SO4 incorporation was unaffected by IGF. Serum promoted the uptake of all three precursors into tissue-bound glycosaminoglycans. It was postulated that IGF could stimulate proteoglycan synthesis not only by elongating existing chondroitin sulphate chains but also by increased synthesis of other sugar chains e.g. keratan sulphate and oligosaccharides.