Subcellular Distribution Of Radioactivity Research Articles

Cell irradiation caused by diagnostic nuclear medicine procedures: dose heterogeneity and biological consequences.

Most radionuclides used for diagnostic imaging emit Auger electrons (technetium-99m, iodine-123, indium-111, gallium-67 and thallium-201). Their very short range in biological tissues may lead to dose heterogeneity at the cellular level with radiobiological consequences. This report describes the dosimetric models used to calculate the mean dose absorbed by the cell nucleus from Auger radionuclides. The techniques used to determine the biodistribution of radiopharmaceuticals at the subcellular level are also described and compared. Published examples of cellular dosimetry computations performed with radiotracers are reviewed in various clinical settings. Finally, the biological implications of the subcellular localization of Auger emitters are examined. While a number of efforts have been made to obtain dosimetric models and to estimate subcellular distribution of radioactivity, little is known of the cellular dosimetry of most radiopharmaceuticals used in diagnostic imaging. However, biological examples of selective radiotracer uptake have been shown, leading to extremely strong cell-cell dose heterogeneity. Furthermore, radiobiological experiments show that the biological effects of Auger emitters incorporated into DNA can be severe, with relative biological effectiveness greater than 1 compared with external X-rays. These findings clearly show that the assessment of biological risks associated with internal administration of diagnostic radiopharmaceuticals must focus not only on target organs as a whole, but also on the cellular level. This review proposes the most appropriate model for dosimetric computations (cellular or conventional) according to the subcellular distribution of radiotracers. The radionuclide of choice and the general strategy used to design new diagnostic radiopharmaceuticals are also discussed.

Cationic Lipids Delay the Transfer of Plasmid DNA to Lysosomes

Plasmid35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time,35S DNA has reached lysosomes. On the contrary, when35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, a lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked35S DNA injection but visible only after more than five hours, when35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

Role of the kidney in the plasma clearance of angiotensinogen in the rat: Plasma clearance and tissue distribution of 125I-angiotensinogen

We studied the tissue distribution and plasma clearance of angiotensinogen (AGN) in rats following an i.v. injection of 125I-labeled AGN. The plasma clearance rate of [ 125I]AGN fits a two-compartment model with half-lives of 10.2 ± 1.5 min and 4.1 ± 0.5 h in non-treated rats and the half-life of slower phase significantly increased to 10.2 ± 1.1 h following bilateral nephrectomy. Radioactivity was predominantly distributed in the kidneys (4.9%) and to a lesser extent in the liver (1.8%), testis (1.2%), spleen (0.61%), heart (0.35%), lung (0.18%), thymus (0.03%) and brain (0.03%). The subcellular distribution of radioactivity in the kidney was 64% in the soluble fraction and 33% in the crude mitochondrial-lysosomal fraction. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the radioactivity in the soluble fraction consisted of proteins corresponding to intact [ 125I]AGN, whereas the mitochondrial-lysosomal fraction contained additional radioactive proteins with molecular weights between 18,000 and 29,000. When isolated kidney cells were incubated with [ 125I]AGN at 0 °C, the radioactive binding was saturable and specific with a Kd value of 4.8 × 10 −11M, whereas incubation at 37 °C resulted in the appearance of degraded products of [ 125I]AGN in the medium. These results suggested that circulating AGN is cleared mainly by the kidneys via receptor-mediated endocytosis, which may play an important role in regulating plasma level of AGN.

Radiolabeled metabolites of proteins play a critical role in radioactivity elimination from the liver

We have recently reported that the behavior of radiolabeled metabolites in the liver appears to be responsible for the hepatic radioactivity levels after administration of protein radiopharmaceuticals. To better understand the role played by radiolabeled metabolites in hepatic radioactivity levels, two benzyl-EDTA derivatives rendering different radiolabeled metabolites, 1-(4-isothiocyanatobenzyl) ethylenediaminetetraacetic acid (SCN-Bz-EDTA) and 1-[ p-(5-maleimidopentyl)aminobenzyl] ethylenediaminetetraacetic acid (ECMS-Bz-EDTA), were selected as bifunctional chelating agents (BCAs), and 111In labeling of galactosyl-neoglycoalbumin (NGA) and mannosyl-neoglycoalbumin (NMA) was performed. Biodistribution of radioactivity in mice and subcellular distribution of radioactivity in hepatocytes were then compared. After accumulation in hepatic parenchymal cells, NGA-EMCS-Bz-EDTA- 111 In rendered a faster elimination rate of radioactivity from the liver than NGA-SCN-Bz-EDTA- 111 In. Although each 111In-NMA exhibited a delayed elimination rate of radioactivity from the liver compared to the 111In-NGA counterpart, NMA-EMCS-Bz-EDTA- 111In showed faster elimination rate of radioactivity than NMA-SCN-Bz-EDTA- 111In. Analyses of radioactivity excreted in feces and urine and remaining in the liver indicated that both BCAs rendered mono-amino acid adducts as the major radiolabeled metabolites (cysteine-EMCS-Bz-EDTA- 111In and lysine-SCN-Bz-EDTA- 111In), which were generated in both cell types of the liver within 1 h postinjection. Subcellular distribution of radioactivity indicated that the radioactivity was copurified with lysosomes. These results demonstrate that although in vivo stability of radiometal chelates is essential, the biological properties of the radiolabeled metabolites generated after lysosomal proteolysis in hepatocytes play a critical role in radioactivity elimination from the liver.

Modelling of the relationship between cell dimensions and mean electron dose delivered to the cell nucleus: application to five radionuclides used in nuclear medicine

The mean dose delivered to the cell nucleus by electron emissions of 99Tcm, 123I, 111In, 67Ga and 201Tl was evaluated at the subcellular level. Models were applied assuming uniform distributions of radioactivity throughout the nucleus, the cytoplasm or the cell membrane, allowing computation of the total absorbed fraction phi and S-values to the cell nucleus as a function of cell dimensions. The graphs of phi plotted according to cell dimensions show that the dose to the cell nucleus strongly depends on the subcellular distribution of radioactivity, the nucleus radius Rnucl and the cytoplasmic thickness e. For a nuclear distribution, phi ranges from 0.1 to 0.35 for the radionuclides studied and S from 0.049 cGy Bq-1 s-1 to 5.503 cGy Bq-1 s-1. In the case of a cell membrane localization, the maximum is obtained for 123I ( phi =0.016). For a cytoplasmic distribution, the maximum is obtained for 201Tl with a value of 0.036. To ease future calculations, third-degree polynomials have been separately fitted to the relationship between the mean absorbed dose to the nucleus for activity accumulated in the nucleus, cytoplasm or surface of the cell membrane. We found a good agreement between our computations and the values obtained by the polynomials. The relative difference between the two methods is always less than 0.7%, 2.8% and 4.5% respectively for nuclear, cell membrane and cytoplasmic distributions.

Binding and retention of estrogen in the uterus of hamsters treated neonatally with diethylstilbestrol

We have shown previously that postpubertal estrogen exposure promotes the development of uterine tumors in hamsters treated neonatally with diethylstilbestrol (DES). The purpose of this study was to determine if the uterine estrogen receptor system of adult hamsters was altered after neonatal DES treatment. There was no effect on the concentration, subcellular distribution, apparent binding affinity, sedimentation properties, surface charge characteristics or ligand specificity of uterine estrogen receptor in ovariectomized, estrogen-replaced animals. Furthermore, neonatal DES treatment had no effect on the subcellular distribution of radioactivity in the uterus of adult animals ovariectomized 1 week before challenge with [ 3H]-estradiol-17β(E 2). However, the amount of radioactivity taken up and specifically bound within the uterus 6 h after [ 3H]-E 2 challenge was less in DES-treated compared to control animals. Six hours after challenge with unlabeled E 2, the concentration (pmol/g tissue) of occupied but not total nuclear estrogen receptor was reduced in DES-treated animals. The difference in uterine radioactivity levels and occupied nuclear receptor retention appears to be due to a difference in estrogen metabolism since the systemic concentration of authentic [ 3H]-E 2 was lower in DES-treated animals compared to control. These results demonstrate no DES-induced change in the physicochemical or functional properties of the uterine estrogen receptor system, suggesting that a lesion in this receptor system is not involved in the etiology of uterine tumor development following neonatal DES exposure. However, the DES-treated animal appears to have an enhanced estrogen metabolism.

Subcellular distribution of human asialotransferrin type 3 in the rat liver.

A small quantity of 125I-labeled human asialotransferrin type 3 (2 to 4 microgram/100 g) was injected in intact rats and the distribution of the hepatic radioactivity analyzed by fractionation of liver homogenates on continuous sucrose density gradient. The ligand rapidly partitioned between plasma membrane and the interior of the cell at an approximate ratio of 1 to 4. The ratio remained constant between 3 min and 1 h. Intracellular 125I was encapsulated in a particle that was of a median equilibrium density of 1.11 (1.109 to 1.114) g/cm3 at 20 degrees C. The ligand recovered from the particles showed no sign of proteolytic digestion and was bound by the immobilized asialoglycoprotein-binding lectin from rabbit liver. The electron microscopic appearance of the subfractions containing of the entrapped ligand closely resembled that of an intermediate Golgi preparation. Various attempts were made to separate the ligand-containing particles from sialyltransferase and phosphodiesterase I activities, but complete separation could not be accomplished. 125I-Asialoorosomucoid studied in the same quantities and under the same conditions as asialotransferrin, yielded a subcellular distribution which was distinct from that of asialotransferrin type 3. Increasing the dose of asialotransferrin, to a level at which rapid catabolism of this asialoprotein occurs, profoundly changed the subcellular distribution of radioactivity. The subcellular distribution thus obtained was comparable with that found for asialoorosomucoid. These findings suggest that asialotransferrin type 3 is associated with different intracellular vehicles (different endosomes?) depending on whether the protein is simply diacytosed or is en route to lysosomes.

The intracellular distribution of high density lipoproteins taken up by isolated rat hepatocytes

The subcellular distribution of 125I-labelled HDL taken up by rat hepatocytes in vivo and in vitro has been studied with subcellular fractionation techniques: differential centrifugation and isopycnic centrifugation in sucrose gradients. 125I-labelled HDL bind to plasma membranes both in vivo and in vitro and part of the membrane-bound 125I-labelled HDL can be dissociated by the addition of unlabelled HDL. The hepatocytes also internalize 125I-labelled HDL. The 125I-labelled HDL accumulate, however, at different intracellular sites in the in vivo and in vitro situation. The subcellular distribution pattern of 125I-labelled HDL taken up by the cells in vivo is similar to that of the lysosomal marker enzyme acid phosphatase. Peak activity was found at a density of 1.20 g/ml. In vitro 125I-labelled HDL accumulate in an organelle with a medium density of about 1.13 g/ml. This distribution was similar to that of the plasma membrane marker 5'-nucleotidase. The subcellular distribution of radioactivity taken up in vivo was changed to lower density by incubating the cells with chloroquine, a drug known to render the lysosomes more boyant. Chloroquine had no effect on the distribution of 125I-labelled HDL taken up by hepatocytes in vitro.

Studies on haem biosynthesis in rat brain.

Abstract— Abnormalities involving haem biosynthesis have been postulated as underlying mechanisms in the aetiology of the neural manifestations of acute porphyria and of lead poisoning. This paper reports a study of the enzymes of the haem biosynthetic pathway and their control in mammalian brain. The activity of rat brain 6‐aminolaevulinate synthetase (ALA synthetase), 6‐aminolaevulinate dehydratase (ALA dehydratase), uroporphyrinogen I synthetase, uroporphyrinogen decarboxylase and ferrochelatase were found to be between 12.5 and 0.002% of the corresponding values for liver. This accords with the lower concentrations of total haem and cytochrome P450 found in brain and with the slower rate of incorporation of [4‐14C]ALA into brain haem in vivo. The subcellular distribution of radioactivity following intraventricular injection of [4‐14C]ALA confirmed that the bulk of brain haemoproteins are intramitochondrial in contrast to liver where the major portion is microsomal. Brain haem biosynthesis was apparently unaffected by factors known to influence this pathway in liver, including starvation and treatment with allylisopropylacetamide or phenobarbitone. These findings suggest that brain haem requirements are considerably less than those of liver and are not subject to significant fluctuations under normal circumstances. Apparent non‐inducibility of ALA synthetase suggests that deficient haem and consequently haemoprotein production could result where other enzymes in the pathway become rate‐limiting due to genetic defects or inhibition by exogenous agents such as lead.

Studies on nitrosamine metabolism I. Subcellular distribution of radioactivity in tumor-susceptible tissues of RFM mice following administration of [ 14C] dimethylnitrosamine

The distribution of radioactivity in tumor-susceptible (liver and lung) and non-tumor-susceptible (heart, forestomach, and esophagus) tissues of male RFM mice was investigated at timed intervals following a single intragastric administration of 14C-labeled DMN. ‡ The greatest amount of radioactivity was associated with the tumor-susceptible tissues—liver and lung. At 15 min, the relative amount of radioactivity in the homogenates of heart, forestomach, esophagus, livers and lung was 1, 2, 3, 10, and 70, respectively. The AS components of lung contained about six times as much radioactivity as the liver 15 min after administration; at 16 hr, the level of radioactivity had decreased and was equal in amount. The AI components of both tumor-susceptible tissues incorporated much less radioactivity than the AS components, indicating that only a small amount of methyl label is covalently bound to cellular macromolecules. The amount or radioactivity in the AI components ranged from 2–34% in the lung and from 11–33% in the liver. In the lung C-fraction the range of radioactivity was 75–89% for the AS components and 52–74% for the AI components. The radioactivity in the AS components of liver C-fraction ranged from 50–89%, and from 52–68% for the AI components. The results suggest differences in the affinity, transport, and/or metabolism of DMN between liver and lung, as well as between tumor-susceptible and non-tumor-susceptible tissues.

Demonstration of nuclear 3H-estradiol binding in hypothalamus and amygdala of female, androgenized-female, and male rats.

The subcellular distribution of radioactivity in female, androgenized female, and male rats 1 hour after the iv administration of tritiated estradiol was examined. High regional specificity among nucle ar fractions of brain areas was seen with the preoptic-anterior hypothal amic area (POA-AH), median eminence-basal hypothalamus (ME-BH), and amygdala having much higher nuclear binding than other brain areas. Nuc lear binding was receptor-mediated in POA-AH, ME-BH, amygdala, dorsal hypothalamus, and the prehypothalamic area in all groups, as demonstrate d by competetive inhibition with diethylstilbestrol (DES). At this time period no differences between male and female rats in DES-blockable radioactivity levels of either nuclear or supernatant fractions in any brain area or pituitary were observed. Androgenized females had lower DES-blockable, nuclear radioactivity levels in POA-AH, ME-BH, and uteri but the % of total tissue radioactivity in the nuclei did not differ between groups for any tissue. These results demonstrate that males and females have similar estradiol uptake systems that are capable of high levels of nuclear binding in various brain areas.

Subcellular distribution and fate of radioactivity in ovaries of pseudo-pregnant rats after in vivo administration of (131-I)HCG.

ABSTRACT After in vivo application of [131I]HCG to pseudo-pregnant rats most of the radioactivity is found in the ovaries. Ovarian homogenate contains per mg protein 50 times as much radioactivity as the liver and 7 times as much as the kidney. The relatively high amount of radioactivity in the kidney possibly reflects a rapid excretion of metabolized or damaged hormone. The subcellular distribution of radioactivity after in vivo application of the labelled hormone indicates a biologically occurring process which cannot be studied by in vitro experiments. Various binding components are shown to exist in the individual subcellular compartments of ovaries which diverge in their binding affinity and capacity for HCG. Binding sites in the nuclear fraction are already saturated when injecting 10 μg HCG together with the label. The uptake of radioactivity in the cytosol, however, is only inhibited when using higher doses of HCG. 1 μg LH-RH provokes maximum release of endogenous LH, inhibits radioactivity uptake by the corpuscular subcellular compartments and does not affect the binding components in the cytosol. It may be concluded that the hormone itself penetrates the cell membrane to reach its target site within the cell or that the recovered radioactivity in the different fractions is due to plasma membrane contamination which may represent the actual hormone binding sites.

The uptake of testosterone and zinc in vitro by the human benign hypertrophic prostate.

SUMMARY The uptake of 65Zn and [1,2-3H]testosterone by minced tissue of human benign hypertrophic prostates and the subcellular distribution of radioactivity were examined. The nature of steroid and 65Zn binding by the cytosol (105000 g supernatant) fraction was investigated by gel filtration, ion-exchange chromatography and electrophoresis. It was found that steroid binding after incubation at 4°C was specific. One or two regions of steroid binding were observed after gel filtration of the cytosol using Sephadex G-200, depending upon incubation conditions. Binding of 65Zn was found in the low molecular weight peak after G-200 gel filtration. Equimolar CdCl2 and 65ZnCl2 were incubated with [1,2-3H]testosterone and minced tissue and the cytosol was subjected to gel filtration. Compared with control values, without CdCl2, reduction of 65Zn binding by about 50% occurred, while binding of 3H-labelled steroid was unaffected. Electrophoresis and ion-exchange chromatography showed that 65Zn and 3H-labelled steroid were bound to different proteins. A sample of the zinc-binding protein was prepared by ion-exchange chromatography and the homogeneity was checked by electrophoresis.

Metabolism of L-DOPA and 5-HTP in the exocrine pancreas studied with autoradiography in the electron microscope

By application of autoradiographic technique in the electron microscope, the cellular and subcellular distribution of radioactivity in the exocrine pancreas of the mouse was investigated after intravenous administration of L-DOPA-3H or dl -5-HTP-3H. Autoradiographic silver grains were recorded over acinar cells, whereas very few grains were found over other cells in the exocrine pancreas. Twenty minutes after administration of 5-HTP-3H autoradiographic grains appeared over all compartments of the acinar cells, but quantitative analyses of autoradiographic sections revealed a concentration of the label to the zymogen granules. This granular preponderance of the labeling was still more marked at 1 hour. Two and 3 hours after injection of 5-HTP-3H, decreasing levels of radioactivity were recorded over zymogen granules. A very similar labeling pattern was obtained when DOPA-3H was injected, but this amino acid had a more rapid turnover. Injection of a decarboxylase inhibitor before the administration of the labeled amino acids abolished the granular concentration of autoradiographic grains, which were now uniformly distributed over all parts of the acinar cells. This observation, together with earlier data, was taken as evidence that most of the silver grains over the zymogen granules represent monoamines formed by decarboxylation of the administered precursors. Pretreatment with an MAO inhibitor before the administration of dl -5-HTP-3H resulted in an increased level of radioactivity compared with animals given only the labeled amino acid.

Quantitative electron microscopic autoradiography on the mouse thyroid gland after administration of 3 H-L-DOPA.

The cellular and subcellular distribution of radioactivity in the mouse thyroid gland different times (20 min — 8 hours) after intravenous administration of 3H-L-DOPA was studied by means of quantitative electron microscopic autoradiography. High concentrations of autoradiographic silver grains occur over parafollicular cells and adrenergic nerves while the labelling of follicular cells and lumina is low or absent and similar to the labelling of connective tissue cells at all observation times. Over the parafollicular cells high levels of radioactivity can be recorded already 20 min after administration of the labelled amino acid. The grain counts are highest at 1 hour and decrease then at 2.5 and 8 hours. The intracellular distribution of label is similar at all observation times; thus, the concentration of silver grains over the typical cytoplasmic granules of the parafollicular cells is 4–5 times higher compared to the concentration over the remainder of the cytoplasm and the nucleus. Treatment with a decarboxylase inhibitor prior to the injection of 3H-L-DOPA results in a low and uniform labelling of all thyroid cells. This finding, taken together with the observation that also pretreatment with reserpine abolishes the autoradiographic reaction over the cytoplasmic granules, gives strong support to the idea that the great majority of silver grains observed over parafollicular cells represents dopamine formed by decarboxylation of the labelled precursor.

Monoamines in the pancreatic islets of the mouse. Subcellular localization of 5-hydroxytryptamine by electron microscopic autoradiography.

By application of autoradiographic technique the cellular and subcellular distribution of radio-activity in mouse pancreatic islets was investigated following intravenous administration of3H-5-hydroxytryptophan. Autoradiographic silver grains, most of which probably represent 5-hydroxytryptamine formed from the labelled precursor, appeared over A2 and B cells, whereas very few grains were recorded over A1 cells at any time investigated (20 min–16 hours) and also when monoamine oxidase was inhibited. Quantitative analysis of autoradiographic sections revealed that the concentration of silver grains over the specific granules of A2 and B cells was 5–10 times higher than over the remaining parts of these cells. In A2 cells the highest grain count was recorded at 20 minutes, in B cells at 1 hour after the injection of label. After 8 hours very few, and after 16 hours no silver grains appeared over islet cells. Inhibition of monoamine oxidase caused an increased retention of label over islet cells, most pronounced over A2 cells. Pretreatment with reserpine abolished the autoradiographic reaction.

Absorption of vitamin B12 by the guinea-pig. II. The subcellular localization of cyano-, methyl- and adenosyl-cobalamins and the effect of fluoroacetate on cyanocobalamin absorption.

Summary.The gastrointestinal distribution and subcellular localization of radioactivity in the ileum of the guinea‐pig was determined 2 hr after oral feeding of physiological doses of cyano‐, methyl‐ and 5′ desoxyadenosyl‐cobalamin. The gastrointestinal distribution of the three analogues was similar although the uptake of methyl‐ and adenosylcobalamin by the ileum was less than that of cyanocobalamin. All three analogues were significantly localized in the mitochondrial fraction of the ileal mucosa during absorption.Guinea‐pigs were injected with sodium fluoroacetate, a drug which is thought to interfere with the conversion of cyano‐ to adenosylcobalamin. Large doses of the drug caused a marked delay in gastrointestinal transit. The subcellular distribution of radioactivity in the ileum was similar in the control group and those pretreated with a small dose (5μg/100 g) of fluoroacetate.It is concluded that the mitochondrial localization of vitamin B12 during absorption by the ileum plays a fundamental role in the transport of the vitamin but that interconversion of cyano‐ to adenosylcobalamin is not the reason for this localization.

Vitamin B 12 in the rat kidney. Evidence for an association with lysosomes

The subcellular distribution of microbiological vitamin B 12 activity and of radioactivity was studied in the rat kidney, 2–22 days after the administration of 20 mμg 58CoB 12. The subcellular distribution of radioactivity was the same as that of the microbiological B 12 at all times. Over 50% of each was in the mitochondrial fraction at a greater concentration than in any other subcellular fraction. Investigation of the distribution of microbiological vitamin B 12 activity and of radioactivity among three mitochondrial subfractions, from 6–29 days after administration of a dose of 57Co- or 58CoB 12, showed the distributions of the two to be the same at all times. Almost 50% of the B 12 was in the lysosome-rich subfraction (M 3) at a concentration per mg protein about six times greater than in any other mitochondrial subfraction or in any of the subcellular fractions. Density-gradient centrifugation confirmed the association of B 12 with lysosomes. The M 3 subfraction was unable to take up 57CoB 12 in vitro. Using an M 3 fraction from a rat previously administered 57CoB 12, it was shown that on incubation at 37 ° radioactivity was released with similar kinetics to the release of lysosomal enzymes.

On the mechanism of hormone action. XV. Subcellular distribution and binding of (1,2-3H)cortisol in rat liver.

Abstract 1. 1. The subcellular distribution of radioactivity in rat liver was investigated 20 min after intraperitoneal injection of 20 and 200 μC of [3H]cortisol per 100 g body wt. The bulk of the liver radioactivity was found in the cytosol fraction and ony 0.2–0.4% was recovered in the purified nuclei. 2. 2. Only 10% of the radioactivity in the cytosol fraction shows RF values similar to that of cortisol in thin-layer chromatography. The major part of this untransformed radioactivity is tightly bound to a macromolecular fraction sedimenting at 4 S. The binding of cortisol to this macromolecule(s) was resistant to ribonuclease but sensitive to proteolytic enzymes and neuraminidase. 3. Virtually all the radioactivity recovered in the salt extracts of purified nuclei was indistinguishable from cortisol by thin-layer chromatography and was found to be weakly associated with a macromolecular fraction in a form resistant to nucleases but sensitive to pronase, trypsin and neuraminidase. At low salt concentrations, this macromolecule(s) shows a sedimentation constant between 8 and 10 S and tends to associate with DNA. AT 0.3 M NaCl the interaction with DNA is weakened and the macromolecule(s) dissociates to give a second component sedimenting at 4 S. 4. 4. The significance of these findings in connection with the initial effect of cortisol on the liver cell, in vivo and in vitro, is discussed.

Differences in the Subcellular Localization of Choline, Acetylcholine and Atropine Taken up by Mouse Brain Slices in vitro

AbstractMouse brain cortex slices have been incubated with tritium labelled acetylcholine, choline and atropine. The subcellular distribution of radioactivity has then been studied by homogenization of the slices in sucrose and separation of the subcellular particles on a continuous exponential sucrose gradient. It was found that about 30 per cent of the radioactivity in the slices were localized in the fraction containing nerve‐endings and mitochondria. Using differential centrifugation and gradient centrifugation of the crude mitochondrial fraction obtained, it was found that acetylcholine is specifically bound to the nerve‐ending particles. Choline had a subcellular distribution similar to acetylcholine, while atropine was not specifically bound to the nerve‐endings.