Synthesis, biodistribution, and autoradiography of radiolabeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689)

35S- and 3H-labeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689) have been synthesized in our laboratory and used to study organ and cellular level distribution in C3H/Km mice bearing RIF-1 tumors. Tissue biodistributions obtained with 35S-WR-3689 showed that blood levels peak at 15 min postinjection and decline gradually over 60 min. At 30 min after drug injection the highest uptake is in kidney and submandibular salivary gland, with lowest levels in brain and moderate to low levels in the RIF-1 tumor, comparable to levels in skin and muscle. High resolution diffusible substance autoradiography with 3H-WR-3689 reveals a homogenous distribution of label over cells in liver and lung and nonuniform distribution of silver grains over the cytoplasm of cells in the kidney cortex, parotid and submandibular salivary glands, and small intestine. There are no indications of preferential nuclear location of label from protective drug in any tissue. Correlations of biodistribution and autoradiography data with measures of radioprotection in different tissues will be useful in interpreting mechanisms of radioprotection with this phosphorothioate.     

Comparison of the protective effects of three phosphorothioate radioprotectors in the RIF-1 tumor

Three aminoalkyl phosphorothioates, WR-2721, WR-3689, and WR-77913, were compared as radioprotectors of RIF-1 tumors irradiated in vivo and assayed for cell survival in vitro. The protector doses were 50% of the acute drug LD50. The radiation dose modifying factors for the three drugs were nearly equal, ranging from 1.5 to 1.7 at surviving fractions of 0.1 and 0.05. Using biodistribution data obtained with 35S labeled drugs, the uptake in tumors was calculated as micromoles drug per gram of tumor. On this basis, tumor levels of WR-77913 were 4.5-fold those of WR-2721, and WR-3689 uptake was 2.7-fold greater than uptake of WR-2721. Thus, on a molar basis, WR-2721 appears to be the most effective protector, but all three phosphorothioates protect this tumor moderately well. In diffusible substance autoradiographs of 3H WR-3689 labeled tumors, label was generally distributed over cells with no evidence of preferential localization over nuclei.     

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.     

WR2721 protection of bone marrow in 131I-labeled antibody therapy.

The use of phosphorothioate radioprotectors such as WR2721 in radioimmunotherapy is attractive because radiation delivered to tumors is usually separated in time from that delivered to the marrow and most normal organs, making protection of tumors of little consequence. However, to be effective radioprotectors must provide continuous protection against radiation of varying dose rates. To evaluate the potential of radioprotectors in radioimmunotherapy we treated normal mice with graded amounts of WR2721 in combination with an LD90/30 (26 MBq) of 131I-labeled antibody. A regimen of 15 doses of WR2721, 200 mg/kg prior to antibody infusion followed by 100 mg/kg ip every 4 h for a total of 72 h, was the maximum tolerated dosage schedule. With this schedule, treatment with radioprotectors failed to prolong survival or delay myelosuppression from the 131I-labeled antibody. In contrast, this regimen of radioprotector provided partial protection from a single treatment of 10 Gy total-body radiation given at 0.2 Gy/min. Protection 30 min after the final dose of WR2721 was greater than 3 h after the 14th dose (60 min prior to the final dose). These results suggest that the potential role of phosphorothioate radioprotectors in a radioimmunotherapy is limited because of the difficulty in achieving continuous protection with nontoxic amounts of drug and possibly because of a limited effect on low-dose-rate radiation.     

Utilization of Plasma Fatty Acid in Rat Brain: Distribution of [14C]Palmitate Between Oxidative and Synthetic Pathways

Radioactivity within individual brain compartments was determined from 5 min to 44 h after intravenous injection of [14C]palmitate into awake Fischer-344 rats, aged 21 days or 3 months. Total radioactivity peaked broadly between 15 min and 1 h after injection, declined rapidly between 1 and 2 h, and then more slowly. In 3-month-old rats, the lipid and protein brain fractions were maximally labeled within 15 min after [14C]palmitate injection, then retained approximately constant label for up to 2 days. Radioactivity in the aqueous brain fraction comprised mainly radioactive glutamate and glutamine, and peaked at 45 min, when it comprised 48% of total brain radioactivity, then decreased to 27% of the total at 4 h, 15% at 20 h, and 10% at 44 h. Percent distribution of radioactivity within the different brain compartments, 4 h after intravenous injection of [14C]palmitate, was similar in 21-day-old and 3-month-old rats, despite higher net brain uptake in the younger animals. The results indicate that about 50% of plasma [14C]palmitate that enters the brain of adult rats is incorporated rapidly into stable protein and lipid compartments. The remaining [14C]palmitate enters the aqueous fraction after beta-oxidation, and is slowly lost. At 4 h after injection, 73% of brain radioactivity is within the stable brain compartments; this fraction increases to 86% by 20 h.     

Radioprotective activity of ethylcellulose microspheres containing WR 2721, after oral administration

Ethylcellulose microspheres containing WR 2721 were prepared by the emulsion-solvent evaporation technique. No significant loss or degradation of this phosphorothioate was noted during preparation. Oral administration of these microspheres to mice gave an important lowering of WR 2721 toxicity and an enhancement of its radioprotective activity with a D.R.F. of about 1.7-1.8 over 2-3 h. This action is explained by the protection of WR 2721 from acid hydrolysis and degradation in the gastro-intestinal tract. The adsorption of a fraction of WR 2721 onto the surface of microspheres constitutes an inconvenience. This study confirms the interest of such carriers for providing important sustained radioprotection after oral administration.     

CELL LOSS AND INFLUX OF LABELED HOST CELLS IN THREE TRANSPLANTABLE MOUSE TUMORS USING [125I]UdR RELEASE

The [¹²⁵I]UdR loss technique was used to estimate cell loss from RIF-1, EMT6 and KHJJ tumors in order to determine the length of the delay between labeling and the beginning of the loss of labeled cells, and also to calculate a value for ø, the cell loss factor. To determine the importance of reutilization of label released from the gut and/or the influx of labeled host cells, the blood flow to some tumors was occluded during and for 30 min after injection of the label. Relatively small amounts of radioactivity entered occluded RIF-1 tumors during 9 days after injection of [¹²⁵I]UdR, indicating that reutilization of systemic label and influx of labeled host cells are not significant in this system. In contrast, substantial amounts of radioactivity entered occluded EMT6 and KHJJ tumors, reaching 40% of the total activity in non-occluded tumors during 6 days following injection. After corrections were made for this influx of label, the [¹²⁵I]UdR loss curves from RIF-1 and EMT6 tumors were essentially exponential from the first day following injection of label. This was interpreted as indicating the loss of proliferating as well as non-proliferating cells from both tumors. The cell loss factor derived from the [¹²⁵I]UdR loss curves corrected for influx appeared to agree well with published values derived from analysis of percent labeled mitoses curves. In contrast, the corrected [¹²⁵I]UdR loss curves from KHJJ tumors showed that loss of activity began three days after injection of label, indicating that primarily nonproliferating cells are lost from this tumor.     

Identity and synthesis of prostaglandins in the lone star tick, Amblyomma americanum (L.), as assessed by radio-immunoassay and gas chromatography/mass spectrometry.

High concentrations of PGE(2) and PGF(2alpha) were identified by radio-immunoassay (RIA) and/or gas chromatography/mass spectrometry (GC/MS) in the hemolymph, salivary glands and saliva of the lone star tick Amblyomma americanum (L.). Binding studies indicated that PGE(2) was free and not bound to any proteins in the hemolymph. A small amount of 6-keto-PGF(1alpha) (breakdown product of PGI(2); prostacyclin) was also found in the salivary glands but not in the hemolymph or saliva. Neither PGD(2) nor PGA(2)/B(2) was detected in any tick material investigated. Although PGE(2) was found in the gut contents, only small amounts of label crossed the gut into the hemolymph during artificial feeding with labeled PGE(2), indicating that the high amounts of PGE(2) in hemolymph and salivary glands are not sequestered from the host blood meal. Isolated salivary glands and salivary gland homogenates demonstrated robust synthesis of PGE(2) at high concentrations of exogenous arachidonic acid. Synthesis by the salivary glands was monitored by measuring increasing PGE(2) with increasing arachidonic acid by RIA, GC/MS and labeled PGE(2) in the presence of labeled arachidonic acid. Synthesis was inhibited in a dose-dependent manner by indomethacin indicating that the cyclooxygenase synthesizing prostaglandins in ticks shares similarities to the enzyme found in mammals.     

Rapid removal to the liver of intravenously injected lipoprotein lipase.

Lipoprotein lipase was purified from bovine milk and labeled with 125I. After intravenous injection to rats the labeled lipase rapidly disappeared from the blood. The initial half-life was about 1 min and more than 70% of the radioactivity was found in the liver at 10 min. 30 min after the injection about 10% of the injected radioactivity was present in acid-soluble form in blood, indicating that the enzyme had been rapidly degraded. Injection of asialofetuin, ribonuclease B or mannan in amounts known to block the hepatic receptors for glycoproteins with exposed galactose, N-acetylglucosamine or mannose residues did not retard the removal of the lipoprotein lipase. Thus, some other, as yet undefined, receptor is implicated. Lipoprotein lipase is known to bind to heparin and some related polysacchrides. Heparin injected before the enzyme delayed its removal and heparin injected after the enzyme caused an immediate increase in blood radioactivity, signifying return from tissues to blood of labeled enzyme. Lipoprotein lipase is present at the endothelium in several extrahepatic tissues and is rapidly turned over. Its presence in blood in appreciable amounts would cause a derangement of lipid transport. The efficient hepatic removal of the enzyme may thus serve an important physiological purpose in keeping the blood levels of this enzyme low.     

Cerebral metabolism of plasma [14C]palmitate in awake,adult rat: Subcellular localization

Following intravenous injection of [U-¹⁴C]palmitate in awake adult rats, whole brain radioactivity reached a broad maximum between 15–60 min, then declined rapidly to reach a relatively stable level between 4 hr and 20 hr. At 44 hr total radioactivity was 57% of the 4 hr value (p<0.05). About 50% of palmitate which entered the brain from the blood was oxidized rapidly, producing¹⁴C-labeled water-soluble components which later left the cytosol. Radioactivity in the cytosolic fraction peaked at 45 min and then declined, coincident with the decline in total brain radioactivity. Membrane fractions were rapidly labeled to levels which remained relatively stable from 1 to 44 hr. Increases in the relative distributions of radioactivity were seen between 1 and 4 hr for the microsomal and mitochondrial fractions, and beyond 4 hr for the synaptic and myelin membrane fractions (p<0.05). Radioactivity in membrane fractions was 80–90% lipid, 5–13% water-soluble components and 3–17% protein. The proportion of label in membrane-associated protein increased with time. Proportions of radioactivity in the combined membrane fractions increased from 65% to 76% to 80% at 4, 20 and 44 hr, respectively. The results show that plasma-derived palmitate enters oxidative and synthetic pathways to an equal extent, immediately after entry into the brain. At and after 4 hr, the radiolabel resides predominantly in stable membrane lipids and protein. Brain radioactivity at 4 hr can be used therefore, to examine incorporation of palmitate into lipids in vivo, in different experimental conditions.     

Quantitative aspects of the uptake and degradation of lysozyme in the rat kidney in vivo

Uptake and degradation of lysozyme in the rat kidney were studied in vivo. The protein was labeled with 125I by way of a moiety (tyramine-cellobiose or 'TC') which remained trapped inside the cells even after proteolysis of the peptide chain (in contrast, the label from conventionally labeled proteins escapes after degradation). Following the injection of 'trapped-label' lysozyme, the radioactivity in the kidneys represented the total amount of lysozyme that was taken up during the experiment. Proteolysis could be followed by determining the amount of acid-soluble degradation products. By adding the radioactivity in the urine to that in the kidneys, a measure of the total filtered load was obtained. When only a trace dose of 125I-labeled TC lysozyme was injected into rats, the amount of radioactivity in the kidneys increased on average by 0.09% per min, after the concentration in the blood had become nearly stable. After 100 min, 30% of the injected dose was recovered in the kidneys. The labeled protein was degraded to acid-soluble molecules of Mr less than 1000. There was apparently a 'lag period' between the endocytosis in the kidneys and the start of degradation. 40 min after the injection of a trace dose, about 0.6% of the 'trapped-label' lysozyme in the kidneys was degraded per min.; subsequently, there was a decline in the fraction which was degraded per min. The amount of lysozyme in the urine increased after the injection of increasing amounts of lysozyme, showing that the capacity of the uptake mechanism was being exceeded, but truly saturating levels of lysozyme could not be reached in vivo.     

Synthesis and migration of proteins and glycoproteins in juxtaglomerular cells of sodium-deficient rats

Summary Sections of juxtaglomerular cells from sodium-deficient rats were subjected to radioautography after a single intravenous injection of L-tyrosine3,5 ³H or of L-fucose ³H to identify the sites of synthesis and to follow the migration of newly-formed proteins and glycoproteins. As early as 2 min after injection of L-tyrosine ³H, the label was highest in the rough endoplasmic reticulum (RER), suggesting that cisternal ribosomes are sites of protein synthesis. By 60 min, much of the label had migrated from the RER to the Golgi complex. Some radioactivity was already present over specific granules by 2 min but a peak was reached at 4h. The label over myofilaments was evident at all time intervals, indicating a certain incorporation of tyrosine into their contractile and/or structural proteins. The label over the cell surface peaked at 4h. After injection of L-fucose ³H, there was an early and important relative specific radioactivity in the Golgi complex at 5 min with a peak at 20 min and a decrease thereafter. The label increased slightly but steadily in secretory granules and cell surface to reach maxima at 4 h. A low level of radioactivity was recorded in mitochondria at all time intervals. After injection of both fucose ³H and tyrosine ³H, the label was detected at relatively low levels in the cytosol. These results suggest that renin, as the major secretory glycoprotein of juxtaglomerular cells, is synthetized in the RER, packaged in the Golgi complex and found relatively rapidly in newly-formed secretory granules. Part of the fucose and tyrosine labels is also associated with the thick cell coat of these cells.Recipient of a summer fellowship from the Kidney Foundation of Canada     

The differential protection by WR2721 of skin versus growing cartilage following irradiation in weanling rats

The potential for radioprotection of growing cartilage by the thiophosphate WR2721 was evaluated in weanling rats using single fractions of irradiation. Protection of acute skin toxicity was monitored simultaneously. Single doses of 600, 1200, 1800, or 2400 cGy were administered to the left tibia of CrL:CD(SD)BR female rats in groups of 12. Identically treated groups were injected with 310 mg/kg WR2721 (2/3 the determined LD50/30) in a concentration of 26 mg/ml intraperitoneally 15 min prior to irradiation. Rats untreated or given WR2721 without radiation served as control groups. Radiographs of the irradiated and unirradiated tibiae for each animal were obtained weekly to the date of sacrifice at 80 days following the initial treatment. Skin toxicity was assessed weekly starting on the second week using Moulder's scale (J.E. Moulder, J.J. Fischer, and A. Casey, Radiology 115, 465-470 (1975]. No significant difference in bone growth as measured by tibial lengths for the WR2721-treated or untreated animals was observed. Skin toxicity including moist desquamation occurred in irradiated limbs and was substantially less in rats treated with WR2721. As opposed to previous work with cysteamine, WR2721 as administered had no significant radioprotective effect on tibial growth in weanling rats but substantially reduced the accompanying skin toxicity.     

WR2721 ameliorates the radiation-induced depression in reactivity of rat abdominal aorta to U46619

Previous studies showed that 20 Gy whole-body gamma irradiation results in a decreased response of the abdominal aorta to the stable thromboxane A2 (TXA2) mimic, U46619. The present study evaluated the effect of WR2721 on this radiation-induced decrease in vascular responsiveness. Rats receiving WR2721 (200 mg/kg, i.p.) 20 min before irradiation showed no depression in vascular reactivity to U46619 compared to control. The abolition of the radiation-induced decrease in vascular responsiveness was not caused by a direct vasoconstrictor action of WR2721 or its metabolites. The vascular response of rat abdominal aortic rings to KCl was unchanged after in vivo exposure to ionizing radiation. WR2721 did not alter the vascular response to KCl. These studies confirm that exposure to whole-body ionizing radiation decreased abdominal aortic vascular responsiveness to U46619. This depressed vascular reactivity can be abolished by pretreatment with the radioprotectant, WR2721. These observations may provide a rapid initial screening method for evaluating the in vivo efficacy of radioprotectant drugs.     

The tissue distribution and the pattern of excretion of [14C]-13-labeled 12, 13-epoxytrichothec-9-ene in mice and rats

The distribution in the mouse tissues of 13-[14C]-12,13-epoxtrichothec-9-ene administered intravenously was determined by whole-body autoradiography and by tracing the radioactivity of the tissues oxidized in an Auto Sample Oxidizer. The appearance of the label in urine and feces was also followed by the tracer technique. The distributions of radioactivity in tissues as determined by the two methods were almost identical. On the autoradiograms of mice killed 10 min after the injection, marked blackening of the film was observed at the sites corresponding to the liver, kidney, and bladder with urine, and much less darkening at other sites. The radioactivities contained in the liver, kidney, urine and small intestine were 13.3, 2.3, 2.6 and 10.2% of the dose, respectively. The labeled toxin was rapidly excreted into urine and feces, 56.0 and 4.9% in 6 hr and 66.7 and 28.0% in 24 hr after injection, respectively. Oral administration of the labeled toxin to mother mice resulted in the appearance of radioactivity in the stomach contents of 7-day suckling mice, thus demonstrating indirectly the secretion of the toxin into the milk. An attempt to show a respiratory route of excretion in rats given the radioactive compound orally or intravenously failed to detect any radioactivity in the expired CO2 collected for 6 hr, suggesting that the 14C in the epoxy ring was intact.     

Protein synthesis in salivary glands of Drosophila melanogaster: relation to chromosome puffs

The salivary glands and other tissues from Drosophila melanogaster were dissected at various times throughout the prepupal period, as well as after heat shocks and ecdysterone treatments, and the proteins labelled by incubating the isolated tissues with [³⁵S]methionine were separated by electrophoresis on sodium dodecyl sulphate-polyacrylamide gel. The labelled band patterns from salivary gland, as seen on the autoradiograph of the gel, showed striking variations, in a manner remarkably similar to variations in puff patterns during the same prepupal period. In proteins from Malpighian tubes, the pattern of bands varied to a lesser extent and in brain only a few components were modified.Heat shock brought about the appearance of a number of new bands, while others were reduced in intensity. This effect was observed with all the tissues examined, salivary glands, brain and Malpighian tubes, as well as wing imaginal discs, tissue lacking polytene chromosomes. The six most heavily labelled bands induced by heat shock represent about 30%, and one component alone represents over 15%, of the total label in the sample, as seen in salivary glands, brain and Malpighian tubes. The synthesis of RNA at puff sites was investigated after heat shock by [³H]uridine labelling. By correlating the amount of [³H]uridine in some puffs with the level of [³⁵S]methionine in some bands a tentative relation is suggested in a few instances.The effect of ecdysterone treatment was also studied in the salivary glands. Changes in a number of protein bands were noticed, though they were much less pronounced than those following heat shock.     

SYNTHESIS, MIGRATION, AND RELEASE OF PRECURSOR COLLAGEN BY ODONTOBLASTS AS VISUALIZED BY RADIOAUTOGRAPHY AFTER [3H]PROLINE ADMINISTRATION

The elaboration of dentin collagen precursors by the odontoblasts in the incisor teeth of 30–40-g rats was investigated by electron microscopy, histochemistry, and radioautography after intravenous injection of tritium-labeled proline. At 2 min after injection, when the labeling of blood proline was high, radioactivity was restricted to the rough endoplasmic reticulum, indicating that it is the site of synthesis of the polypeptide precursors of collagen, the pro-alpha chains. At 10 min, when the labeling of blood proline had already declined, radioactivity was observed in spherical portions of Golgi saccules containing entangled threads, and, at 20 min, radioactivity appeared in cylindrical portions containing aggregates of parallel threads. The parallel threads measured 280–350 nm in length and stained with the low pH-phosphotungstic acid technique for carbohydrate and with the silver methenamine technique for aldehydes (as did extracellular collagen fibrils). The passage of label from spherical to cylindrical Golgi portions is associated with the reorganization of entangled into parallel threads, which is interpreted as the packing of procollagen molecules. Between 20 and 30 min, prosecretory and secretory granules respectively became labeled. These results indicate that the cylindrical portions of Golgi saccules transform into prosecretory and subsequently into secretory granules. Within these granules, the parallel threads, believed to be procollagen molecules, are transported to the odontoblast process. At 90 min and 4 h after injection, label was present in predentin, indicating that the labeled content of secretory granules had been released into predentin. This occurred by exocytosis as evidenced by the presence of secretory granules in fusion with the plasmalemma of the odontoblast process. It is proposed that pro-alpha chains give rise to procollagen molecules which assemble into parallel aggregates in the Golgi apparatus. Procollagen molecules are then transported within secretory granules to the odontoblast process and released by exocytosis. In predentin procollagen molecules would give rise to tropocollagen molecules, which would then polymerize into collagen fibrils.     

Radioautographic studies on radiosulfate incorporation in the digestive organs of mice.

The sulfate uptake and accumulation in mouse digestive organs were studied by light microscopic radioautography. Two litters of normal ddY mice 30 days after birth, each consisting of 3 animals, were studied. One litter of animals were sacrificed 30 min after the intraperitoneal injections with phosphate buffered Na2(35)SO4, and the other litter animals were sacrificed 12 hr after the injections. Then several digestive organs, the parotid gland, the submandibular gland, the sublingual gland, antrum and fundus of the stomach, the duodenum, the jejunum, the ileum, the caecum, the ascending colon and the descending colon were taken out. The tissues were fixed, dehydrated, embedded in epoxy resin, sectioned, picked up onto glass slides, coated with radioautographic emulsion by a dipping method. AFter the exposure, they were developed, stained with toluidine blue and analyzed by light microscopy. As the results, many silver grains were observed on serous cells of the salivary glands, mucosa and submucosa of the stomach, villous cells and crypt cells of the small intestines and whole mucosa of the large intestines at 30 min after the injection. Then at 12 hr after the injection silver grains were observed on mucous cells of the salivary glands, some of the stomach glands, and mucigen granules of goblet cells in the small intestines and the large intestines. The numbers of silver grains observed in respective organs at 30 min were less than those at 12 hr. From these results, it is concluded that glycoprotein synthesis was demonstrated in several digestive organs by radiosulfate incorporation. In the salivary glands the silver grains were more observed in serous cells at 30 min, while in mucous cells more at 12 hr than 30 min after the injection. In other organs the silver grains were more at 30 min than at 12 hr. These results show the time difference of glycoprotein synthesis in respective organs.     

Biosynthesis of beta nerve growth factor in mouse submaxillary glands

The biosynthesis of beta nerve growth factor (betaNGF) was studied in isolated mouse submaxillary glands incubated with L-[35S]cystine. Sodium dodecyl sulfate gels of anti-betaNGF immunoprecipitates from labeled gland homogenates showed a single major peak of radioactivity, which comigrated with purified betaNGF. This species was nearly completely precipitated by the addition of equivalent amounts of anti-betaNGF, but was absent from immunoprecipitates obtained by the addition of ferritin plus anti-ferritin. The cystine-containing tryptic peptides of the labeled species appeared identical with those of purified betaNGF. In submaxillary glands from adult male mice, labeling of betaNGF represented approximately 0.2% of the trichloroacetic acid-precipitable radioactivity. Castration reduced this value to one-third, while testosterone treatment of castrated animals restored the relative betaNGF synthesis to normal or more. No betaNGF synthesis could be detected in glands from female animals. Several tissues were examined for their ability to synthesize betaNGF in culture. Only submaxillary gland incorporated detectable amounts of radioactivity into betaNGF. Labeling of betaNGF could also be obtained by direct injection of isotope into the submaxillary gland in vivo. The results are discussed in terms of the integration of betaNGF synthesis into neuronal development and maintenance.     

Suppression of plasma thyroid hormone concentrations by cortisol in the European eel Anguilla anguilla

Female European eels, Anguilla anguilla, were given a single intra-arterial injection via a catheter of cortisol hemisuccinate at doses ranging from 3.5 to 35 micrograms (15 to 150 micrograms/kg body wt), yielding mean plasma cortisol levels of 87-410 ng/ml 2 hr after injection. Cortisol treatment (17.5 and 35 micrograms) significantly decreased plasma levels of thyroxine (T4) and triiodothyronine (T3) within 24 hr relative to those in control fish. Cortisol treatment (35 micrograms) appeared to increase the clearance rate of 125I-T3 from plasma and the proportionate uptake of radioactivity in certain tissues after injection of 125I-T3. Cortisol treatment had no apparent effect on the plasma clearance of 125I-T4 or tissue distribution of radioactivity after injection of 125I-T4.