Effects of 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one on the synthesis of cholesterol and ubiquinone in rat intestinal epithelial cell cultures

The relationship between cholesterol and ubiquinone synthesis in rat intestinal epithelial cell cultures was examined by using 3 beta-[2-(diethylamino)ethoxy]androst-5-en-17-one hydrochloride (U18666A). Addition of U18666A to cells caused a greater than 90% inhibition of incorporation of [3H]acetate into cholesterol and an apparent large increase in the incorporation of [3H]acetate and [3H]mevalonate into ubiquinone. However, the incorporation of 4-hydroxy[U-14C]benzoate, a ring precursor of ubiquinone, was unchanged. The apparent increase of 3H incorporation into ubiquinone was found to be due to the formation of a contaminant that has been identified as squalene 2,3:22,23-dioxide. Following incubation of cells with U18666A, its removal from the medium resulted in a decrease in squalene 2,3:22,23-dioxide labeling and a corresponding increase in the polar sterol fraction. These results demonstrate that U18666A inhibits the reaction catalyzed by 2,3-oxidosqualene cyclase (EC 5.4.99.7). As a result, the isoprenoid precursors are diverted not to ubiquinone as has been suggested but to squalene 2,3:22,23-dioxide, a metabolite not on the cholesterol biosynthetic pathway. Removal of the drug allows cyclization of squalene 2,3:22,23-dioxide, leading to formation of compounds with chromatographic properties of polar sterols.     

Selective Inhibition of Cholesterol Biosynthesis in Brain Cells by Squalestatin 1

Abstract: The effect of squalestatin 1 (SQ) on squalene synthase and other enzymes utilizing farnesyl pyrophosphate (F-P-P) as substrate was evaluated by in vitro enzymological and in vivo metabolic labeling experiments to determine if the drug selectively inhibited cholesterol biosynthesis in brain cells. Direct in vitro enzyme studies with membrane fractions from primary cultures of embryonic rat brain (IC₅₀ = 37 n M ), pig brain (IC₅₀ = 21 n M ), and C6 glioma cells (IC₅₀ = 35 n M ) demonstrated that SQ potently inhibited squalene synthase activity but had no effect on the long-chain cis -isoprenyltransferase catalyzing the conversion of F-P-P to polyprenyl pyrophosphate (Poly-P-P), the precursor of dolichyl phosphate (Dol-P). SQ also had no effect on F-P-P synthase; the conversion of [³H]F-P-P to geranylgeranyl pyrophosphate (GG-P-P) catalyzed by partially purified GG-P-P synthase from bovine brain; the enzymatic farnesylation of recombinant H-p21 ^ras by rat brain farnesyltransferase; or the enzymatic geranylgeranylation of recombinant Rab1A, catalyzed by rat brain geranylgeranyltransferase. Consistent with SQ selectively blocking the synthesis of squalene, when C6 glial cells were metabolically labeled with [³H]mevalonolactone, the drug inhibited the incorporation of the labeled precursor into squalene and cholesterol (IC₅₀ = 3–5 µ M ) but either had no effect or slightly stimulated the labeling of Dol-P, ubiquinone (CoQ), and isoprenylated proteins. These results indicate that SQ blocks cholesterol biosynthesis in brain cells by selectively inhibiting squalene synthase. Thus, SQ provides a useful tool for evaluating the obligatory requirement for de novo cholesterol biosynthesis in neurobiological processes without interfering with other critical reactions involving F-P-P.     

Cholesterol for Synthesis of Myelin Is Made Locally, Not Imported into Brain

Abstract: We examined whether cholesterol needed for myelin formation is locally synthesized or whether it comes from the circulation. The experimental design was to inject [³H]water and to use incorporation of label into brain cholesterol as a measure of the rate of accumulation of newly synthesized cholesterol in brain. The contribution of the circulation to this labeled cholesterol pool was minimized by repressing liver synthesis of cholesterol with a high cholesterol diet. The rate of accumulation of total cholesterol was calculated from the increasing amounts of sterol in brain regions at successive time intervals during development. Thus, accumulating cholesterol not explained as being newly synthesized (radioactive) could be assumed to have come from the circulation. Long-Evans rats, ranging in age from birth to 35 days, were injected intraperitoneally with [³H]water (0.3–1.0 mCi/g of body weight) and killed 2 h later. The brain was dissected into brainstem, cerebellum, and cerebral hemispheres, and total lipids were extracted. Cholesterol and its precursors were quantified by HPLC. The radioactivity associated with the sterol fractions and the specific activity of body water determined from serum were used to calculate the absolute amount of newly synthesized sterol. The rates of cholesterol synthesis were compared with the rates of accumulation of total cholesterol in each brain region. The rate of accumulation of total sterol (cholesterol and desmosterol) closely followed that of newly synthesized total sterol in all brain regions from the second through the fifth postnatal weeks. Thus, all sterol accumulation in brain during the period of rapid myelination can be explained by local synthesis; neither diet nor production of cholesterol by other organs plays a direct role in supplying cholesterol for myelination in brain.     

Brain-to-blood elimination of 24S-hydroxycholesterol from rat brain is mediated by organic anion transporting polypeptide 2 (oatp2) at the blood-brain barrier

24S-Hydroxycholesterol (24S-OH-chol), a major cerebral cholesterol metabolite, is an endogenous ligand for the liver X receptor and is a potential stimulant of cholesterol release from glial cells. The elimination mechanism of 24S-OH-chol from the brain is one of the key issues for understanding cerebral cholesterol homeostasis. The purpose of the present study was to clarify the molecular mechanism of the elimination process of 24S-OH-chol across the blood–brain barrier (BBB). After an intracerebral injection in rats, [³H]24S-OH-chol was eliminated from the brain and the radioactivity derived from [³H]24S-OH-chol was detected in the plasma, while [³H]cholesterol was not significantly eliminated from the brain. Co-administration of unlabeled 24S-OH-chol significantly inhibited the [³H]24S-OH-chol elimination, while no inhibitory effect was seen at the same concentration of cholesterol. The [³H]24S-OH-chol elimination was inhibited by co-administration of probenecid, but not by benzylpenicillin. Pre-administration of digoxin completely inhibited the elimination. Xenopus laevis oocytes expressing rat oatp2 exhibited significant transport of [³H]24S-OH-chol, and this was inhibited by unlabeled 24S-OH-chol and digoxin, indicating that rat oatp2 transports 24S-OH-chol. These results are the first direct demonstration that 24S-OH-chol undergoes elimination from the brain to blood across the BBB via a carrier-mediated process, which involves oatp2 expressed at the BBB in rats.     

Regulation of squalene synthetase in human hepatoma cell line Hep G2 by sterols, and not by mevalonate-derived non-sterols

Incubations of Hep G2 cells for 18 h with human low-density lipoprotein (LDL) resulted in a decrease of squalene synthetase activity, whereas heavy high-density lipoprotein (hHDL) stimulated the activity. Simultaneous addition of LDL abolished the hHDL-induced stimulation, indicating that manipulating the regulatory sterol pool within the cells influenced the enzyme activity. Blocking the endogenous cholesterol synthesis either at the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase site with compactin or at the 2,3-oxidosqualene cyclase site with the inhibitor U18666A gave rise to an elevation of the squalene synthetase activity. Simultaneous addition of mevalonate abolished the compactin-induced increase. However, at total blockade of sterol synthesis by 30 microM U18666A, added compactin and/or mevalonate did not change the enzyme activity further. It was concluded that sterols regulate the squalene synthetase activity, whereas, in contrast with the regulation of the HMG-CoA reductase activity in Hep G2 cells, mevalonate-derived non-sterols did not influence this enzyme.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

The Effects of Insect Juvenile Hormone on the Growth of Some Protozoans in vitro. I. Crithidia sp.

SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10⁻⁵M-10⁻³M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10⁻³M) inhibited the incorporation of [³H]thymidine, [³H]uridine and [³H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [³H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10⁻³M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane.     

[3H]Xylamine Accumulation by Two Sympathetically Innervated Tissues

Abstract: The accumulation of [³H]xylamine ([ring-G-³H]- N -2'-chloroethyl- N -ethyl-2-methylbenzylamine; [³H]XYL) by rabbit thoracic aorta and rat vas deferens was studied in vitro . [³H]XYL uptake in both tissues saturated at the same concentrations and displayed the same time course as that for maximal inhibition of [³H]noradrenaline ([³H]NA) accumulation by XYL. Hydrolysis of [³H]XYL or coincubation with Na₂S₂O₃ reduced tissue accumulation of tritium by 80%. In aorta and vas deferens, about 45% and 65%, respectively, of the total [³H]XYL accumulation was blocked by 100 μmol/L desmethylimipramine (DMI), l -NA, or bretylium. In the absence of sodium ion, the uptake of [³H]XYL was reduced by approximately these same amounts. In both tissues nearly 70% of the [³H]XYL taken up was protein bound when measured as trichloroacetic acid-precipitated tritium. Of this radioactivity, the same proportion was sensitive to 100 μmol/L DMI or the absence of sodium ion as found for total tissue accumulation of [³H]XYL. Hydrolysis of [³H]XYL prior to incubation with vas deferens reduced the protein-bound tritium by more than 95%. The studies indicate that [³H]XYL interacts with NA uptake carrier in both rabbit aorta and rat vas deferens and that a substantial portion of the resulting protein-bound radioactivity is carrier-dependent.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

Uptake of [3H]Dopamine into Dopaminergic and Noradrenergic Neurones of the Isolated Neurointermediate Lobe of the Rat Hypophysis. Effects of Desipramine and Nomifensine

Abstract: The isolated neurointermediate lobe (NIL) of the rat hypophysis accumulates [³H]dopamine from the incubation medium. Column chromatographic analysis showed that 92% of the tissue radioactivity was contained in the catecholamine fraction. [³H]Dopamine represented 70% and [³H]noradrenaline 30% of the [³H]catecholamines. Desipramine (1 μM) prevented the formation of [³H]noradrenaline without affecting the storage of [³H]dopamine. Nomifensine (10 μM) blocked the storage of [³H]dopamine and [³H]noradrenaline. Thus, in the NIL, [³H]dopamine is taken up into dopaminergic and noradrenergic neurones. In the latter, [³H]dopamine is converted to [³H]noradrenaline, indicating a significant dopamine β-hydroxylase activity in the NIL tissue. A selective labeling of the dopamine stores with [³H]dopamine can be achieved in the presence of desipramine.     

Rate of Incorporation of R3hlthymidine In Various Tissues of the Mouse A basis for the evaluation of genotoxic effects of chemicals

Abstract. [³H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible.
The present paper deals with some practical aspects on the use of [³H]thymidine in vivo. [6-³H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol Were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and dNA-associated radioactivities. A rapid increase of the [³H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities asśociated with DNA closely eorrelated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [³H]thymidine incorporation into DNA.     

Mobilisation of reserves and the earliest synthesis of sterols and latex triterpenes during germination and early seedling growth of Euphorbia lathyris

Seedlings of Euphorbia lathyris L. were grown in the dark at 25°C. Levels of amino acids, sugars and soluble phosphate in the endosperm increased upon germination after 4 days of imbibition, while the amounts of mineral reserves Mg²⁺ and K⁺ started decreasing. Ca²⁺ was not translocated from the endosperm to the seedling. Maximum values for amino acids were found on day 7, and the highest amounts of sugars were present on day 10. The endosperm was completely depleted by day 12.
Before germination (days 1–3) a low level of sterol synthesis in the embryo was detected with labeled sucrose and serine and to a lesser extent with labeled pyruvate. The label of [2-¹⁴C]-mevalonate proceeded exclusively to squalene. Laticifers started the synthesis of their triterpenes upon germination (day 4), using sucrose as a main substrate. A concurrent increase of sterol synthesis outside the laticifers was traced with labeled serine. Radioactive triterpenes, 4 α-methylsterols and sterols were detected in growing seedlings after [¹⁴C]-mevalonate uptake, but most of its label accumulated in squalene. The use of labeled mevalonate in sterol synthesis in growing seedlings is discussed.     

Biosynthesis of squalene and sterols by rat aorta

The synthesis of nonsaponifiable compounds from radioactive mevalonate by segments of adult rat aorta was studied in vitro. The labeled products consisted largely of substances with the chromatographic and chemical behavior of squalene, lanosterol, lathosterol, and cholesterol. Even after 3 or 4 hr of incubation, the incorporation of mevalonate into squalene was higher than its incorporation into C(27) sterols; cholesterol contained less than 20% of the radioactivity in the total sterols. Lanosterol was the most highly labeled sterol. The level of radioactivity in lathosterol was comparable to the level in cholesterol. Small amounts of radioactivity were found in other sterols. Material with the same mobility on TLC as 7-dehydrocholesterol had less radioactivity than cholesterol, but more than sterols with the mobility of desmosterol. The results of measurements made after short periods of incubation showed that squalene and lanosterol became labeled before the other nonsaponifiable compounds.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in human hepatoma cell line Hep G2. Effects of inhibitors of cholesterol synthesis on enzyme activity.

Incubating Hep G2 cells for 18 h with triparanol, buthiobate and low concentrations (less than 0.5 microM) of U18666A, inhibitors of desmosterol delta 24-reductase, of lanosterol 14 alpha-demethylase and of squalene-2,3-epoxide cyclase (EC 5.4.99.7) respectively, resulted in a decrease of the HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase activity. However, U18666A at concentrations higher than 3 microM increased the HMG-CoA reductase activity in a concentration-dependent manner. None of these inhibitors influenced directly the reductase activity in Hep G2 cell homogenates. Analysis by t.l.c. of 14C-labelled non-saponifiable lipids formed from either [14C]acetate or [14C]mevalonate during the cell incubations confirmed the sites of action of the drugs used. Beside the 14C-labelled substrates of the blocked enzymes and 14C-labelled cholesterol, another non-saponifiable lipid fraction was observed, which behaves as polar sterols on t.l.c. This was the case with triparanol and at those concentrations of U18666A that decreased the reductase activity, suggesting that polar sterols may play a role in suppressing the reductase activity. In the presence of 30 microM-U18666A (sterol formation blocked) the increase produced by simultaneously added compactin could be prevented by addition of mevalonate. This indicates the existence of a non-sterol mevalonate-derived effector in addition to a sterol-dependent regulation. LDL (low-density lipoprotein), which was shown to be able to decrease the compactin-induced increase in reductase activity, could not prevent the U18666A-induced increase. On the contrary, LDL enhanced the U18666A effect, showing that the LDL regulation is not merely the result of introducing cholesterol to the cells.     

Insensitivity of Ubiquinone Biosynthesis in Glioblastoma Cells to an Epileptogenic Drug, U18666A

Abstract: To investigate the perturbation of ubiquinone biosynthesis by a hypocholesterolemic drug, 3β-(2-di-ethylaminoethoxy)androst-5-en-17-one hydrochloride (U18666A), we measured the incorporation of radioactive mevalonate, methionine, tyrosine, and 4-hydroxybenzoic acid into ubiquinone in glioblastoma cells. These four precursors unanimously showed that ubiquinone biosynthesis was not significantly altered by U18666A, which blocked cholesterol biosynthesis at steps beyond mevalonate formation. The fluctuation of the endogenous mevalonate level had little effect on ubiquinone biosynthesis, implying the relative stability of cellular ubiquinone biosynthesis. Furthermore, exogenously added mevalonate did not have an appreciable effect on ubiquinone biosynthesis. The major ubiquinone produced in rat glioblastoma cells was identified as ubiquinone-9. The mevalonate-derived products accumulated in the U18666A-treated cells differed significantly from those reported in a broken cell study, suggesting the existence of delicate mechanisms regulating the formation of cholesterol intermediates.     

Chloroquine inhibits cyclization of squalene oxide to lanosterol in mammalian cells

Chloroquine inhibits the incorporation of [14C]acetate into sterols at a concentration of 10 microM or more in mouse L cells but has no effect on fatty acid synthesis and CO2 production from the same substrate even at a 10-fold higher concentration of the drug. The site of inhibition is distal to the formation of mevalonate since chloroquine also inhibits [14C]mevalonate metabolism to sterols and does not decrease the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) or the incorporation of [14C]acetate into the total nonsaponifiable lipids. Analyses by thin layer and high pressure liquid chromatography of the nonsaponifiable lipid fraction from cultures incubated with chloroquine show an accumulation of radioactivity in the region of squalene oxide. Identification of the radiolabeled lipid as squalene oxide has been established by: (a) its co-migration with the authentic squalene oxide standard; (b) its conversion into squalene glycol by acid hydrolysis; and (c) its further metabolism to desmosterol when chloroquine is removed from the medium. Addition of chloroquine (12.5-50 microM) to 20,000 X g supernatant fractions of mouse liver homogenates inhibits the incorporation of [14C]mevalonolactone into cholesterol and lanosterol, with corresponding increases of [14C]squalene oxides, in a concentration-dependent manner. It appears, therefore, that chloroquine inhibits the enzymatic step catalyzed by 2,3-oxidosqualene-lanosterol cyclase (EC 5.4.99.7). Incubation of cell cultures with chloroquine (50 microM) arrests cell growth and causes cell death after 1-3 days. However, simultaneous incubation of chloroquine with either cholesterol or lanosterol prevents cell death and permits cell growth. Uptake of chloroquine is not affected by exogenous sterols since intracellular chloroquine concentrations are the same in cells grown with or without added sterols. The cytotoxicity of chloroquine, under our experimental conditions, must, therefore, be due primarily to its inhibition of sterol synthesis. In addition to its well known effect on protein catabolism, chloroquine has been found to inhibit protein synthesis. The significance of these findings concerning the use of chloroquine in studying the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity is discussed.     

DEMONSTRATION OF ACETYLCHOLINE RELEASE BY MEASURING EFFLUX OF LABELLED CHOLINE FROM CEREBRAL CORTICAL SLICES

Abstract— To demonstrate release of ACh in the absence of inhibition of cholinesterase, slices of cerebral cortex were incubated with [³H]choline, after which they were placed in a tissue bath for superfusion. Hemicholinium (HC-3) increased the spontaneous efflux of [³H]choline. Electrical stimulation at 4/s increased the efflux of [³H]choline to the same extent whether the slices were stimulated early or late during superfusion. The effect of stimulation on efflux of [³H]choline was abolished by tetrodotoxin and by the absence of calcium. The extent of choline efflux resulting from stimulation, as calculated from the specific radioactivity of the incubation medium, was the same when the slices were incubated with 0.1 or 1.0mM choline, but was less with lower concentrations of choline. We conclude that the increased efflux of [³H]choline evoked by stimulation probably originates from stores of [³H]ACh synthetized during incubation.     

[3H]thymidine labels less than half of the DNA-synthesizing cells in the mouse tumour, carcinoma NT

Abstract. We have studied carcinoma NT, a transplantable mouse adenocarcinoma of spontaneous origin. Cells labelled with [³H]thymidine ([³H]TdR) were restricted to a narrow zone around the periphery of this tumour and were also found in rings up to 50 μ m wide, around isolated blood vessels in the central necrotic area. Labelling with [³H]deoxyuridine ([³H]UdR), another DNA synthesis precursor, produced a very different pattern. The labelled zone around the periphery was much wider than with [³H]TdR, and [³H]UdR labelled cells were found up to 110 μ m from isolated vessels. [³H]iododeoxyuridine ([³H]IUdR) gave the same pattern of labelling as [³H]UdR. In the heavily labelled zone, within 1 mm of the tumour periphery, the labelling index (LI) was 51% after [³H]UdR or [³H]IUdR injection, and only 36% with [³H]TdR.
The data show that at least half of the DNA-synthesizing cells in this tumour did not incorporate [³H]TdR. Previous workers reported cell loss factors for carcinoma NT of 60% calculated from [³H]TdR labelling data and 30% from the rate of loss of [¹²⁵I]UdR. The present work suggests that calculations based on [¹²⁵I]UdR data are more likely to be accurate for carcinoma NT than those using [³H]TdR data.     

Kinetics of Entry of P0 Protein into Peripheral Nerve Myelin

Abstract: Sciatic nerves from 9-day-old rat pups were removed, sliced into 0.4-mm sections, and incubated with [³H]fucose or [¹⁴C]glycine precursors. The nerve slice system gave nearly linear incorporation of [³H]fucose as a function of time for 3 h, after an initial lag of ˜30 min for homogenate and ˜60 min for myelin. Incorporation of [³H]fucose at constant specific radioactivity was directly proportional to exogenous fucose levels over the range 3.0 × 10⁻⁸ m to 1.5 × 10⁻⁶ m . Analysis of labeled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that greater than 50% of labeled glycoprotein was P₀, with no other major constituents. This system was used in fucose-chase experiments to determine that a period of ˜20 min elapses between fucosylation and assembly of P₀ into myelin. Cycloheximide inhibition of protein synthesis was used to determine that a period of ˜33 min elapses between protein synthesis and appearance of P₀ myelin.