EFFECT OF STRESS ON THE DISPOSITION OF CATECHOLAMINES LOCALIZED IN VARIOUS INTRANEURONAL STORAGE FORMS IN THE BRAIN STEM OF THE RAT

Abstract— The utilization of [³H]norepinephrine newly taken up or newly synthesized from [³H]tyrosine was studied in the brain stem of normal and stressed rats up to 5 h after the intracistemal injection of [³H]norepinephrine or [³H]tyrosine. The biphasic disappearance of the exogenous as well as of the endogenously synthesized [³HJnorepinephrine revealed that the amine is localized in at least two main compartments (A and B). The half-life of the amine newly taken up or newly synthesized, mainly localized in compartment A, is of short duration (between 15 and 30 min); the amine stored for a longer period of time and mainly distributed in compartment B is utilized more slowly (half-life, 180 to 260 min). A stress of short duration (15 min) induced by electric shocks applied to the feet increased the utilization of [³HJnorepinephrine newly taken up or newly synthesized from [³H]dopamine or [³H]tyrosine, but has no effect on the [³H]norepinephrine stored for a longer time period, indicating that the amine in compartment A is released in preference to that stored in compartment B. A stress of longer duration (180 min) increased the utilization of [³H] norepinephrine in both compartments and induced a sustained increased in norepinephrine synthesis as shown by the enhanced formation of [³H]norepinephrine from [³H]tyrosine in brain stem slices in vitro. The electrical stress was without effect on [³H]norepinephrine uptake. As for [³H]norepinephrine, the 15 min of stress enhanced the utilization of [³H] dopamine newly taken up or newly synthesized from [³H]tyrosine and had no effect on [³H]dopamine stored for a longer time period. These results suggest an increased release of both [³H]dopamine and [³H]norepinephrine from noradrenergic terminals of the rat brain stem. Finally, the 15 min of stress appeared to have no effect on the utilization of [³H] serotonin newly synthesized from [³H]tryptophan in serotonergic neurons of the brain stem, whereas the 180 min of stress increased the utilization of 5-HT in this structure.     

IN VITRO EXPERIMENTS ON THE METABOLISM, ACCUMULATION AND RELEASE OF 5-HT IN THE NERVOUS SYSTEM OF THE SNAIL HELIX POMATIA

Abstract— The accumulation, metabolism and stimulated-induced release of 5-HT in the nervous system of the snail was studied. When nervous tissue was incubated at 24°C in a medium containing [¹⁴C]5-HT or [³H]tryptophan, tissue: medium ratios of about 25:1 and 4:1 respectively were obtained after 45 min incubation. The process responsible for [¹⁴C]5-HT accumulation showed properties of an active transport system: it was temperature sensitive and was greatly inhibited by dinitrophenol and ouabain. Furthermore, the accumulation process was inhibited by imipramine and desipramine. Of a number of analogues of indole, N-acetyl-5-HT and 5-hydroxytryptophan were the most potent in the inhibition of the accumulation of [¹⁴C]5-HT. The presence of a large molar excess of amino acids had little effect. A small amount (less than 14 per cent) of the accumulated [¹⁴C]5-HT was metabolized to form 5-hydroxyindole acetic acid, even after long periods (2 h) of incubation. The accumulated [³H]tryptophan was metabolized to form 5-hydroxytryptophan and 5-HT; the content of formed [³H]5-HT increased with incubation time whilst the [³H]5-hydroxytryptophan remained more or less constant. The presence of p-chlorophenylalanine in the incubation medium did not interfere with the accumulation of [³H]tryptophan, though it inhibited the formation of [³H]5-hydroxytryptophan and to a greater extent [³H]5-HT. A rapid efflux of the accumulated [¹⁴C]5-HT from snail nervous tissue was observed on electrical stimulation. Slower release resulted when the Ca²⁺ ion content of the incubation medium was replaced by Mg²⁺ ions. There is also a slight efflux of radioactive substances following electrical stimulation in tissues previously incubated in [³H]tryptophan. Most of this radioactivity was attributed to the formed [³H]5-HT. The data support the idea that 5-HT is a transmitter-substance in the snail Helix pomatia, and that re-uptake of the substance is a method of inactivating the released amine.     

MONOAMINES AS POSSIBLE MEDIATORS IN THE REGULATION OF FAST AXOPLASMIC FLOW

—The effect of drugs which have been shown to alter monoamines in the CNS on the rate of fast axoplasmic flow of [³H]leucine labelled material in the rat sciatic nerve was examined. Drugs which deplete monoamines led to a decrease while those which increase the level of monoamines led to an increase in the rate of fast axoplasmic flow. Monoaminergic drugs were also found to alter the amount of [³H]leucine incorporated into spinal cord.     

THE NUCLEAR PERMEABILITY, INTRACELLULAR DISTRIBUTION, AND DIFFUSION OF INULIN IN THE AMPHIBIAN OOCYTE

[³H]Inulin (mol wt ≈ 5,500) solutions are microinjected into the cytoplasm of mature oocytes of Rana pipiens and the subsequent movement of the solute recorded by quantitative ultralow temperature autoradiography. The autoradiographs show transient cellular diffusion gradients, the influence of the nucleus on these gradients, and the nuclear:cytoplasmic distribution of inulin. Analysis leads to the following conclusions: (a) Inulin diffuses in cytoplasm at about 3 x 10^-6 cm²/s, or one-fifth as rapidly as in water. Most of this decrease is attributable to the increased tortuosity of the diffusional path due to the presence of inclusions and macromolecules. (b) The nuclear envelope is very permeable to inulin; its resistance to inulin's passage is similar to that of cytoplasm. The envelope appears to play a negligible role in regulating the nucleocytoplasmic movement of solutes smaller than macromolecules, (c) Inulin concentrates in the nucleus to four times its cytoplasmic level; this is attributed to solute exclusion from cytoplasmic water. Evidence is presented that among hydrophilic solutes the degree of exclusion increases with molecular size. The potential significance of cytoplasmic exclusion processes to understanding secretion and the intracellular movement of macromolecules is briefly discussed.     

THE INFLUENCE OF INJECTED TRITIATED THYMIDINE ON THE MITOTIC CIRCADIAN RHYTHM IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH*

The initial effect of an injection of TdR-5-³H (1 μCi/g body weight; 6 Ci/mmol) in the cheek pouch epithelium of the Syrian hamster is an increase in the mitotic index. The increase is observed 1–5 hr after injection, depending upon the time of day when the injection is given, and is followed by compensatory variations in mitotic index. This deviation from the normal circadian rhythm in the mitotic index appears to depend on the fraction of G₂-cells at the time of injection. The main effect is a shortening of t_g2. No effect is observed after injection of non-radioactive TdR or isotonic saline. The results of the present experiment emphasize that unexpected results may be obtained when using mitotic indices from animals labelled with ³H-TdR, as well as the risks of using the PLM-method in a partially synchronized system.     

AN ADAPTATION OF THE PUSH-PULL CANNULA METHOD TO STUDY THE IN VIVO RELEASE OF [3H]DOPAMINE SYNTHESIZED FROM [3H]TYROSINE IN THE CAT CAUDATE NUCLEUS: EFFECTS OF VARIOUS PHYSICAL AND PHARMACOLOGICAL TREATMENTS

Abstract— The release of [³H]dopamine ([³H]DA) continuously synthesized from l-[3,5–³H]tyrosine from the caudate nucleus of the cat was estimated in halothane anaesthetized or‘encéphale isolé’animals. For this purpose, an improved superfusion cannula, avoiding tissue damage, was used. The best localization for the tip of the superfusion cannula was found first by determining the topographical distribution of endogenous DA within the caudate nucleus. A rostro-caudal heterogenous distribution of the transmitter was detected. In perfusion experiments, l-[3,5–³H]tyrosine was introduced continuously at a rate of 33μl/min. [³H]DA was the only catecholamine found in serial 15 min fractions as revealed by cochromatography. The spontaneous release of [³H]DA was greater in anaesthetized than in ‘encéphale isolé’ cats; it represented 150 and 100 times the blank value, respectively. Depolarization by K⁺ (30 mm) applied locally in the striatum or by electrical or mechanical stimulation of the substantia nigra caused a transitory increase in [³H]DA release. Conversely, a decrease in nerve activity induced by tetrodotoxin (5 × 10^?-⁷ m) or by electrocoagulation of the substantia nigra was associated with a decline in the amounts of [³H]DA in superfusates. A temporary reduction in [³H]DA release could also be obtained by a short-lasting cooling block of the substantia nigra. As expected, d-amphetamine (10^?-⁵ m) and benzotropine(10^?-⁷ m) added to the superfusing medium increased [³H]DA release. These pharmacological results, as well as the changes in [³H]DA release observed after various manipulations of the activity of dopaminergic neurones, confirms the validity and the high sensitivity of this approach.     

RADIOAUTOGRAPHIC VISUALIZATION OF THE INCORPORATION OF GALACTOSE-3H AND MANNOSE-3H BY RAT THYROIDS IN VITRO IN RELATION TO THE STAGES OF THYROGLOBULIN SYNTHESIS

Rat thyroid lobes incubated with mannose-³H, galactose-³H, or leucine-³H, were studied by radioautography. With leucine-³H and mannose-³H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-³H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-³H and mannose-³H, but has no detectable effect on galactose-³H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-³H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-³H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.     

SOLUBILITY OF MUSTARD GAS [BIS (β-CHLOROETHYL) SULFIDE] IN WATER,MOLAR SODIUM CHLORIDE,AND IN SOLUTIONS OF DETERGENTS

1. The solubility of mustard (H) in water and in molar sodium chloride was found to be 5.8 x 10^–3 molar and 3.2 x 10^–3 molar respectively or 0.92 mg. per ml. and 0.5 mg. per ml. Solubility curves have been drawn and the usefulness of this method in examining the homogeneity of H preparations as well as in establishing their solubility, is discussed. 2. Certain detergents increase the solubility of H in water. The solubility was found to increase with the concentration of detergent. 3. Many detergents were found to affect the interfacial tension between H and water so that with slight agitation liquid H breaks up into minute droplets. This in turn greatly accelerates the rate of solution.     

THE PERMEABILITY OF THE AMPHIBIAN OOCYTE NUCLEUS, IN SITU

Ultralow temperature radioautography, suitable for the quantitative localization of diffusible solutes, was used to study the permeability of the nuclear envelope in the intact amphibian oocyte Sucrose-³H solutions were injected into mature oocytes, in volumes of 0 016–0 14% of that of the cell, and the subsequent movement of the solute was recorded. The resultant radioautographs show diffusion gradients in the cytoplasm and nucleus, and concentration gradients across the nuclear envelope Analysis of these gradients discloses that the nuclear envelope is as permeable as a comparable structure composed of cytoplasm, and is about 10⁸ times more permeable than the oocyte plasma membrane The diffusion coefficient of sucrose in cytoplasm is 2 x 10^-6 cm²/sec, or about one-third its diffusivity in pure water. This reduction can probably be accounted for by an effective lengthening of the diffusional path because of obstruction by cytoplasmic inclusions. The nuclear: cytoplasmic sucrose concentration ratio at diffusional equilibrium is about 3 05, or 1.6 times as great as expected from the water content of the two compartments This asymmetry is attributed to an unavailability of 36% of the cytoplasmic water as solvent Finally, sucrose entry into oocytes from a bathing solution was monitored by whole cell analysis and radioautography. These and the microinjection results are consistent with a model in which sucrose entry into the cell is entirely limited by the permeability of the plasma membrane. The results are inconsistent with cell models that hypothesize a short-circuit transport route from the extracellular compartment to the nucleus, and with models in which cytoplasmic diffusion is viewed as limiting the rate of solute permeation.     

THE INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

DEVELOPMENT OF THE UPTAKE AND STORAGE OF L-[3H]NOREPINEPHRINE IN THE RAT BRAIN

The uptake and storage of L-[³H]norepinephrine at various stages of development was examined in homogenates of rat brain. For the adult animal, active uptake accounted for 80 per cent of the total uptake. At 14 days of gestation, no active uptake was demonstrable At 18 days of gestation, saturable uptake of L-[³H]norepinephrine with a K_m of 3 × 10 ^?7m was first demonstrable; the K_m value did not vary during subsequent development. The V_max. of uptake increased five-fold between 18 days of gestation and 28 days postnatally, at which stage it was the same as the adult value. The development of saturable uptake paralleled but preceded the increase in endogenous norepinephrine. When homogenates were incubated with l -[³H]norepinephrine and subjected to centrifugation on linear sucrose gradients, there was a peak of tritium in the synaptosomal fractions; the magnitude of the peak increased with maturation of the brain. The increase in the peak of tritium paralleled the increase in particulate LDH activity and was distinct from the peak of MAO activity. Desipramine, a compound that blocks the initial uptake of norepinephrine, first exhibited inhibition of uptake at 19 days of gestation; the degree of inhibition did not vary during subsequent development. In contrast, reserpine, a compound which inhibits the intra-neuronal storage of norepinephrine, exhibited a progressive increase of inhibition with maturation of the brain at and subsequent to 19 days of gestation.     

pH AND POPULATION DENSITY IN THE REGULATION OF ANIMAL CELL MULTIPLICATION

Sparse and dense cultures of chick embryo cells were affected differently by pH. The rates of cell multiplication and of thymidine-³H incorporation into DNA of dense cultures were increased as the pH was increased from 6.6 to 7.6. At pH higher than 7.6 the rate of multiplication decreased slightly in the dense cultures, but the rate of thymidine-³H incorporation continued to increase. The discrepancy was due in part to cell death and detachment at very high pH, and in part to a more rapid uptake of thymidine-³H at very high pH. Sparse cultures were much less sensitive to pH reduction and, when a suitably conditioned medium was used to minimize cell damage, very sparse cultures grew almost as well at pH 6.7 as at higher pH. The rates of cell multiplication and thymidine-³H incorporation at low pH decreased in the initially sparse cultures before they reached confluent cell densities. There was no microscope evidence of direct contact between plasma membranes of cells at these densities although the parallel orientation indicated that the cells were influencing locally each other's behavior. Even at much higher cell densities, electron microscopy revealed large intercellular gaps partly filled with a fragmentary electron-opaque material suspected to be glycoprotein. Wounding experiments showed that pH affected cell migration in a manner similar to its effects on cell multiplication. Low pH inhibited cell migration, but those cells which migrated into the denuded region multiplied as rapidly at low pH as at high pH. The effects of pH on growth were correlated with effects on the uptake of 2-deoxyglucose-³H. Dense populations of cells inhibited by low pH were stimulated to incorporate thymidine-³H by the addition of small amounts of diethylaminoethyl-dextran. Rous sarcoma cells at high cell density were less sensitive to pH than were normal cells at the same density, but were more sensitive than sparse normal cultures. The results suggest that cell growth is inhibited through the combined effects of both lowered pH and high cell density on cell surface permeability.     

EFFECTS OF DOPAMINERGIC AGONISTS AND ELECTRICAL STIMULATION OF THE MIDBRAIN RAPHÉ ON THE RELEASE OF 5-HYDROXYTRYPTAMINE FROM THE CAT BRAIN IN VIVO

—The cerebroventricular system of spinal-sectioned cats was perfused with artificial cerebrospinal fluid after the intraventricular administration of [³H]tryptophan, and the perfusate was analyzed for [³H]5-HT. At the end of the experiment the relative contents of [³H]5-HT in regions lining the cerebroventricular system (caudate nucleus, septum, hypothalamus) were essentially the same as the endogenous 5-HT contents. Electrical stimulation of the midbrain raphé did not alter the efflux of total radioactivity but significantly increased the efflux of [³H]5-HT. The addition of potassium to the perfusing CSF also increased the efflux of [³H]5-HT, while additions of d-amphetamine, apomorphine or l -DOPA were without effect.     

THE SITE OF SYNTHESIS OF GANGLIOSIDES IN THE CHICK OPTIC SYSTEM

Abstract– In the retinas of 1-day-old chickens that received an intraocular injection of N-[³H]acetylmannosamine the labelling of N-acetylneuraminic acid and CMP-N-acetylneuraminic acid increased for at least 8 h and that of gangliosides for at least 24 h after injection. In the optic tectum contralateral to the injected eye at 8 h after the intraocular injection, the labelling of gangliosides exceeded the labelling of gangliosides in the ipsilateral tectum by approx 20-fold. In the contralateral tectum the highest concentration of labelled gangliosides was in subfractions enriched in synaptosomes and synaptic plasma membranes. No significant contralateral ipsilateral differences were found in the acid soluble substances of the tectum. In the optic tectum, labelled gangliosides appeared earlier in the neuronal perikarya than in synaptosomes when the injection was intracranial. Conversely, when the injection was intraocular the labelling appeared earlier in the synaptosomes than in the neuronal perikarya. The radioactivity pattern of the optic tectum gangliosides resembled the pattern of retina gangliosides when N-[³H]acetylmannosamine was injected intraocularly, but when N-[³H]acetylmannosamine was given intracerebrally the radioactivity pattern resembled that of optic tectum gangliosides. Intraocular injection of colchicine or vinblastine did not affect the labelling of retinal gangliosides from N-[³H]acetylmannosamine injected into the same eye but prevented the appearance of labelled gangliosides in the optic tectum. In vitro the ganglioside glycosylating activity of optic tectum synaptosomes and synaptic plasma membranes was between 6 and 10-fold lower than that found in the optic tectum neuronal perikarya. These findings support the notion that the main subcellular site of synthesis of neuronal gangliosides is in the neuronal perikarya, from which they are translocated to the nerve endings.     

STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

SYNTHESIS OF RNA IN NEURONS OF THE HYPOGLOSSAL NERVE NUCLEUS, AFTER SECTION OF THE AXON, IN MICE

Synthesis of RNA in neurons of the hypoglossal nerve nucleus after axonal section was studied by means of [5-³H]uridine administration and radioautographic counting techniques in mice. The results of the experiments were evaluated by counts of silver grains over the nucleoplasm and cytoplasm of the neurons. RNA synthesis was greater in neurons after axonal section, and this increase was evident from 12 hr after the operation. The greatest increases in the operated side were observed in the 1st, 2nd and 3rd days after operation. In the 7th and 14th days RNA synthesis was still greater in the hypoglossal nucleus of the sectioned nerve but the difference in the control nucleus was not so striking. In the 30th day synthesis of RNA in left and right hypoglossal nuclei was comparable.     

CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.     

IDENTIFICATION OF A PROTEIN FRACTION IN THE OCCIPITAL CORTEX OF THE MONKEY RAPIDLY LABELLED DURING THE EXPOSURE OF THE ANIMAL TO RHYTHMICALLY FLICKERING LIGHT

Monkeys exposed to a rhythmically flickering light (flicker frequency 7/sec, intensity 1614 lumens/m²) show a higher incorporation of intracisternally administered l -(U-³H)-lysine into proteins of the visual cortex as compared to monkeys kept in darkness. An increase in specific radioactivity is noticed in both the soluble and particulate (including membrane linked) proteins. The 105,000 g supernatant proteins from the visual cortex have been fractionated on DEAE-cellulose columns followed by resolution of each fraction on polyacrylamide gels. The results suggest that there is a group of acidic low molecular weight proteins whose synthesis is significantly stimulated during the exposure of the animal to flickering light. The fractions give immunological cross-reaction with anti S-100 Serum.     

RAPID AXONAL TRANSPORT IN VITRO IN THE SCIATIC SYSTEM OF THE FROG OF FUCOSE-, GLUCOSAMINE- AND SULPHATE-CONTAINING MATERIAL

—An in vitro system from the frog has been used to study fast axonal transport of glycoproteins. The migration of [³H]fucose-, [³H]glucosamine- and [³⁵S]sulphate-labelled material was followed from the dorsal ganglia, along the sciatic nerve towards the gastrocnemius muscle. The distribution in different subcellular fractions, effect of cycloheximide and transport kinetics did not differ very much between fucose- and glucosamine-incorporation into the nerve. Cycloheximide blocked the synthesis of TCA-insoluble radioactivity, which was transported at a rate of 60–90 mm per day at 18°C, more effectively than the synthesis of stationary proteins in the ganglia. About 10 per cent of the TCA-insoluble and transported radioactivity was extracted by chloroform-methanol (2:1, v/v) and might be glycolipids and the rest glycoproteins. Results suggest that TCA-soluble activity, which was recovered in the nerve, originated in part from labelled macromolecules consumed along the axons. The rapidly transported TCA-insoluble radioactivity was 85 per cent particulate and mainly associated with structures sedimenting in the microsomal fraction. [³⁵S]Sulphate-labelled TCA-insoluble material was resistant towards chloroform-methanol (2:1, v/v) extraction and rapidly transported from the ganglia into the nerve. The synthesis was inhibited by cycloheximide. The material, probably proteoglycans, represented a quantitatively minor part of transported glycoproteins.     

ON THE QUESTION OF LONG-LIVED RNA IN MAMMALS

RNA was isolated from various mouse tissues 126 days after the injection of ¹⁴C-uridine and 37 days after the injection of ³H-uridine. Calculations based on a small but definite residual amount of ¹⁴C-labeled RNA suggest the existence of an RNA with a half-life in excess of 10 days. Independent calculations based on the ratio of ³H and ¹⁴C dpm in chromatographically isolated UMP suggest a minimum half-life of about 30 days.