Suppression of murine lymphocyte proliferation induced by a small RNA purified from theTaenia solium metacestode

A substance fromTaenia solium metacestodes that decreases lymphocyte proliferation induced by concanavalin A was isolated. The molecular weight of this substance was estimated to be slightly more than 1,450 Da. Crude metacestode factor was fractionated through a Bio-gel P-6 column. Peak 1 showed suppressive activity. After incubation with RNase the substance lost its activity. Incubation of this material with trypsin or papain increased its suppressive activity. It was stable at boiling temperature for 10 min. The incubation of this substance with murine macrophages had no effect on [³H]-thymidine uptake by cocultured fresh splenic lymphocytes stimulated with concanavalin A. Conversely, cocultures of lymphocytes pretreated with the substance and fresh splenic lymphocytes showed a decreased incorporation of [³H]-thymidine. These results suggest that this substance is a RNA-peptide molecule whose RNA moiety accounts for its suppressive activity. The findings also suggest that in vivo the factor may be a modulator of the immune response.     

Choline uptake, acetylcholine synthesis and release by Limulus abdominal ganglia

Isolated ganglia from the ventral nerve cord of the horseshoe crab, Limulus polyphemus, were incubated in [³H]choline and subsequently analyzed for choline uptake, conversion of choline to acetylcholine and the release of the newly synthesized acetylcholine. The ganglia readily accumulated radioactivity when incubated in Chao's solution containing 2 μM [³H]choline. The rate of uptake was 0.08 pmols/min/mg tissue and 26% of the [³H]choline taken up during a 1 h period was converted to [³H]acetylcholine. A 15 min exposure of ganglia to 90 mM K⁺ prior to incubation in [³H]choline caused an 89% increase in choline uptake and a 150% increase in its conversion to [³H]acetylcholine. The presence of unlabeled acetylcholine in the uptake medium inhibited both uptake and conversion of [³H]choline.There was a 5-fold increase in the efflux of radioactivity when ganglia incubated in 2 μM [³H]choline were superperfused with 90 mM K⁺. The increased efflux of radioactivity was Ca²⁺-dependent and was inhibited by Mg²⁺ (44%) and by Co²⁺ (72%). Similarly, addition of veratridine caused a Ca²⁺ and Na⁺-dependent release of radioactivity from prelabeled ganglia. Analysis of the superperfusate revealed that virtually all of the released radioactivity was [³H]acetylcholine.The abdominal ganglia of Limulus take up choline at micromolar concentrations, convert substantial amounts of it to acetylcholine and possess a depolarization-triggered, Ca²⁺-requiring mechanism for the specific release of acetylcholine. These results give further support to the view that the abdominal ganglia of Limulus contain a population of cholinergic terminals.     

Homologous and heterologous immunization against the metacestodes ofTaenia saginata andTaenia taeniaeformis in cattle and mice

Summary Cattle and mice were immunized against infection withTaenia saginata andTaenia taeniaeformis, respectively, using antigens obtained from both homologous and heterologous species of cestodes. Mice were protected against infection withT. taeniaeformis when they were immunized intramuscularly or orally with either a somatic antigen extracted from the metacestodes or an excretory/secretory (E/S) antigen collected during the in vitro culture of oncospheres ofT. taeniaeformis. Also, the intramuscular or oral immunization of mice with the E/S antigens from the oncospheres ofT. saginata was highly effective in inducing protection against infection withT. taeniaeformis, as was intramuscular immunization with a somatic antigen extracted from the metacestodes ofTaenia crassiceps and prior infection with viable metacestodes ofT. crassiceps. Furthermore, three-month-old calves developed a protective immunity against infection withT. saginata when they were immunized intramuscularly with the E/S products of oncospheres of the homologous parasite or a heterologous parasite,T. taeniaeformis. In addition, the E/S products of the heterologous parasite,T. taeniaeformis, as well as the homologous parasite,T. saginata, were highly effective when used to immunize pregnant heifers, either intramuscularly or via the intramammary route, resulting in a passive transfer of immunity againstT. saginata to newborn calves.     

Tunicamycin does not inhibit transport of phosphatidylinositol toXenopus rod outer segments

Summary Tunicamycin inhibits the dolichol pathway forN-linked glycosylation of proteins, including photoreceptor opsin, and causes a buildup of tubulo-vesicular profiles in the intersegmental space between photoreceptor rod inner and outer segments associated with disruption of new disc assembly. We tested the hypothesis that a tunicamycin lesion in photoreceptors would block lipid transport into the outer segment. AdultXenopus retinas were preincubated in dim red light with 20 g ml^–1 of tunicamycin for one hour followed by incubation in the light for 2–6 h with tunicamycin plus either [³H]mannose, [³H]leucine, [2-³H]glycerol or [³H]myo-inositol. Tunicamycin caused accumulation of tubulo-vesicular membranes in the intersegmental space and significantly reduced both [³H]leucine and [³H]mannose incorporation into the basal region of rod outer segments. However, tunicamycin had no effect on [³H]glycerol incorporation into the rod outer segment phospholipids. After 5 h incubation with [³H]glycerol, radiolabel in outer segment fractions was associated primarily with phosphatidylinositol in both control and tunicamycin treated retinas. Quantitative light microscope autoradiography of both [³H]glycerol and [³H]inositol labelled retinas showed diffuse labelling over the entire rod outer segment in both control and tunicamycin treated retinas with no accumulation of radioactivity in the basal discs of control retinas or in the tubulo-vesicular structures in the intersegmental space of tunicamycin treated retinas. Our results indicate that despite the morphological disruption and inhibition of glycoprotein transport to outer segments after tunicamycin treatment, transport of labelled phosphatidylinositol occurs normally. These data add to a growing body of evidence separating the lipid and protein transport pathways to the outer segment.     

Intracellular uptake and degradation of extracellular tracers in mouse skeletal muscle in vitro: The effect of denervation

Innervated and chronically denervated mouse skeletal muscles have been incubated under various conditions in a Ringer solution containing one of the three macromolecules: [³H]α-neurotoxin, [³H]inulin and horseradish peroxidase. Following extensive wash-out for 4 h of the extracellular compartment, the amount of each macromolecule retained intracellularly was obtained.Intracellular uptake of a [³H]monoacetylated α-neurotoxin in vitro at 37°C was found to be increased in denervated mouse extensor digitorum longus muscles compared to innervated control muscles. Similarly, the uptake in vitro at 37°C of [³H]inulin and horseradish peroxidase was also increased in denervated muscles. At 4°C the uptake of [³H]inulin and horseradish peroxidase was markedly reduced. Protamine was found to stimulate the uptake of [³H]inulin at 37°C but not at 4°C. Reduction in specific activity by addition of 50-fold excess of unlabelled inulin failed to affect the uptake of [³H]inulin suggesting that this uptake process obeyed bulk kinetics. Furthermore, the endocytized [³H]inulin was found to be strongly retained in the muscles since prolonged washing or addition of unlabelled inulin to the washing solution did not reduce the uptake.Characterization of [³H]inulin taken up by the muscles was performed by gel chromatography on Sephadex G-25. Using a purified [³H]inulin solution it was observed that about 45% of the total radioactivity remaining in the muscles was eluted as [³H]inulin. Additional radioactivity consisted of lower molecular weight compounds. These degradation products of [³H]inulin were only present in the muscle homogenate and were not detected in the incubation solution.The results suggest that intracellular uptake of different macromolecules by endocytosis in skeletal muscles increases following denervation, and that following uptake, degradation of the endocytized material may occur.     

In vitro tumor cell destruction by syngeneic mouse macrophages: Methods for assaying cytotoxicity

Various methods for assaying in vitro cytotoxicity mediated by mouse macrophages were studied. A conventional visual cellular counting procedure was compared to two different radioactive monitoring assays where tumor target cells were prelabeled with either [³H]thymidine or [¹²⁵I] iododeoxyuridine. The radioactive monitoring methods were found to be superior to the visual assay with regard to accuracy, reproducibility and ease of operation, and labeling with [¹²⁵I]iododeoxyuridine surpassed labeling with [³h]thymidine. The results of the macrophage mediated cytotoxicity tests were similar and followed in the same direction with all methods of assay.Target cell destruction by immune syngeneic macrophages occurred at ratios exceeding 40 macrophages to one tumor cell after 5 days of incubation. In assays utilizing target cells prelabeled with radioactive compounds, cytotoxicity was monitored by either the release of radioactivity into the media or by radioactivity remaining in viable adherent target cells. Reutilization of radioactive label by macrophages was not demonstrable. It appears that exact quantitative determination of macrophage mediated cytotoxicity in vitro by radioactive monitoring offers a simplified and reproducible method of assay.     

A comparison of pre- and postsynaptic effects of alpha-adrenolytic drugs in the pulmonary artery of the rabbit.

Strips of the main pulmonary artery of the rabbit were preincubated with [³H]noradrenaline and then superfused and electrically stimulated transmurally. Yohimbine, dihydroergotamine and tolazoline at low concentrations enhanced the stimulation-evoked overflow of tritium; up to one hundred times higher concentrations were needed in order to reduce stimulation-evoked contractions. Piperoxan and mianserin enhanced the evoked overflow and reduced the evoked contractions at identical concentrations. Phentolamine, azapetine, clozapine and phenoxybenzamine at low concentrations diminished the contractile response; up to thirty times higher concentrations were needed to increase the evoked overflow of tritium. At high concentrations, all drugs accelerated basal tritium outflow. Separation of individual [³H]-compounds revealed that a high concentration of piperoxan markedly accelerated the basal outflow of [³H]3,4-dihydroxyphenylglycol. Lower concentrations of piperoxan and yohimbine that did not affect the basal efflux of [³H]-compounds greatly increased the evoked overflow of [³H]noradrenaline as well as of [³H]3,4-dihydroxyphenylglycol and [³H]normetanephrine.The pulmonary artery contains postsynaptic α-adrenoceptors which mediate smooth muscle contractions. Its noradrenergic neurones possess presynaptic α-receptors which mediate inhibition of the per pulse release of noradrenaline by a negative feedback mechanism. α-Adrenolytic drugs vary widely in their relative pre- and postsynaptic effects. Yohimbine, dihydroergotamine and tolazoline preferentially block presynaptic α-receptors and at low concentrations selectively facilitate noradrenaline release. Phentolamine, azapetine, clozapine and phenoxybenzamine preferentially block postsynaptic α-receptors and at low concentrations selectively antagonize the contractile response. Piperoxan and mianserin occupy an intermediate position. In a given tissue, pre- and postsynaptic α-receptors may differ in their structure.     

Selective bidirectional transport of [3H]d-aspartate in the pigeon retino-tectal pathway

Selective uptake and bidirectional axonal migration of tritiated D-aspartate ([³H]d-asp) within a sub-population of retino-tectal neurons was shown by radioautography in the pigeon visual system. Six hours after either pulse injection or prolonged topical application of [³H]d-asp to the optic tectum, light microscope radioautographs exhibited an intense and preferential labeling of incoming optic fibers, within tectal layer 1. These axons could be traced back to their perikarya in the ganglion cell layer of the contralateral retina.Conversely, 6–48 h after intra-vitreous injection of [³H]d-asp, anterogradely-labeled axons could be followed from the fiber layer of the retina to layer 1–7 of the contralateral optic tectum. In addition, several mesodiencephalic primary visual relay nuclei showed some degree of radioautographic labeling. Labeled retinofugal axons originated from a restricted contingent of small intensely reactive ganglion cells which were heterogeneously distributed throughout the retina. These cells were particularly numerous in the postero-superior quadrant which is known to project to the ventro-caudal aspect of the tectum. Indeed, both radioautography and liquid scintillation measurements showed that the bulk of anterogradely transported radioactivity (more than 80% as free asp) was accumulated in this tectal region. In contrast, when [¹⁴C]l-leucine was injected together with [³H]d-asp, anterogradely transported¹⁴c-labeled proteins were evenly distributed throughout the tectum.Finally, part of the radioactivity present in tectum 48 h after intraocular [³H]d-asp could be released ‘in vitro’ by 47 mm K⁺ stimulation, in a Ca²⁺ dependent manner.These results strongly suggest that the pigeon retinal ganglion cells which are here reported selectively to take up, transport and release [³H]d-asp may well utilize l-aspartate, l-glutamate or a closely related substance as a neurotransmitter.     

Single-step selection of mammalian cell mutants deficient in CTP synthetase

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS ^–)is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [³H]uridine and [³ H]thymidine. The CTPS ^– mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS ^– cells are deficient in CTP synthetase and thus are unable to convert [³ H]UTP into [³ H]CTP, which eventually is converted into [³ H]dCTP and incorporated into DNA; (2) the growth of CTPS ^– mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [³ H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 × 10 ^–7.The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [³ H]uridine labeling assay. This modified tritium suicide method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.     

Biosynthesis of [7-3H]16α-hydroxy-dehydroepiandrosterone having high specific activity

Biosynthesis of [7-³H]16α-hydroxy-dehydroepiandrosterone in high specific activity has been studied. [7-³H] dehydroepiandrosterone (13.9 C/mM) in trace quantity was oxidized by Streptomyces roseochromogenes (NRRLB-1233) for 5 min at 27 °C. The radioactive products were chromatographically separated, identified and their radiochemical purity established by isotopic dilution analysis. [7-³H]16α-hydroxy-dehydroepiandrosterone (2.5 × 10⁷ dpm) was obtained by microbial hydroxylation of substrate (1.9 × 10⁹ dpm). In some cases [7-³H]5-androstene-3β, 16α, 17β-triol in a small amount of radioactivity could be found at the prolonged reaction for 30 hr.     

The effects of N-acetylated alpha-linked acidic dipeptidase (NAALADase) inhibitors on [H]NAAG catabolism in vivo

N-Acetylated, alpha-linked acidic dipeptidase (NAALADase) is a chloride-activated, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-aspartyl-glutamate (NAAG) in vitro. To determine whether NAALADase is the catabolic enzyme of NAAG in vivo, we have examined the effects on [³H]NAAG metabolism of intrastriatal co-injections of agents that affect NAALADase activity in vitro. Co-injections of NAALADase inhibitors, such as quisqualate (Quis), phosphate, dithiothreitol and EGTA were found to prolong the of [³H]NAAG, whereas cobalt, a NAALADase activity stimulator, accelerated [³H]NAAG catabolism. These results are consistent with a role for NAALADase in the extracellular disposition of endogenous NAAG.     

Labelling of schwann and satellite cells by [3H]dexamethasone in a rat sympathetic ganglion and sciatic nerve

Light-microscopic autoradiograms of superior cervical ganglia of adult rats after injection of [³H]dexamethasone showed a preferential accumulation of radioactivity in the nuclei of satellite and Schwann cells. The total radioactivity found in the ganglion at 10 min after injection was high compared to the cerebral cortex of the brain, but declined rapidly. Only Schwann and satellite cell nuclei retained the label for up to 2 h. The labelling was specific for dexamethasone, since it could be prevented by an excess of unlabelled dexamethasone but not by corticosterone. Administration of [³H]corticosterone resulted in only a weak labelling of the ganglion and no specific labelling of Schwann and satellite cells could be observed. As in the ganglion, accumulations of [³H]dexamethasone by Schwann cell nuclei was also observed in the sciatic nerve.It is possible, therefore, that satellite and Schwann cells represent an important target for glucocorticoids in the nervous system.     

Radio-labelling of 1-O-alkyl, 2-O-acetyl,sn-glycero-3-phosphorylcholine, 1-O-(9,10-di3H)-octadecyl PAF-acether

³H-labelling of 1-³H-PAF-aceter was prepared by catalytic reduction of 1-O-(9,10)-octadecenyl-2-O-benzyl-sn-glyceryl-3-phosphorylcholine and then acetylated. 5 moles of this dehydro derivative dissolved in methanol was treated during 4 hours with tritium gas (80 Ci) and compressed until 1.1 bar by an automatic tritium transfer unit [1]. The catalyst was removed by filtration over Millex (Millipore) and labile tritium atoms eliminated by rotatory flash evaporation. The chemical purity was checked by³H scanning and autoradiography. It was finally recovered through preparative thin-layer chromatography and then acetylated. The specific radioactivity was found to be close to 40–50 Ci/mmole.     

Identification and measurement of rat eosinophil phospholipase D. Its activity on schistosomula phospholipids

A sensitive assay, using [¹⁴C]lecithin as a substrate, has been developed for the measurement of phospholipase activity in rat peritoneal polymorphonuclear leukocytes. Cell extracts were found to contain a phospholipase D activity and indirect evidence suggested that eosinophils are responsible for the cleavage of lecithin. Intact peritoneal cells were also able to hydrolyze exogenous [¹⁴C]lecithin in vitro. When [³H]choline-labeled schistosomula were used as targets in antibody-dependent cytotoxicity experiments, the radioactivity of lecithin decreased more rapidly in a complete cytotoxicity system than in controls, suggesting that hydrolysis of schitosomula phosholipids occured during the killing process.     

Regulatory role of dopa and components of the cyclic AMP system

Injection of nonradioactive dopa (1 mg/mouse) before injection of [³H]dopa (20 Ci/mouse) into albino mice with Harding-Passy melanoma leads to increased accumulation of tritium in the tumor tissue. Radioactivity of the melanoma in this experimental group was twice as high as the radioactivity of the tumor tissue in animals receiving injections of [³H]dopa alone. Investigation of adenylate cyclase and phosphodiesterase activity and the cyclic AMP level in the melanoma of the mice 2 h after injection of dopa (1 mg/mouse) revealed accumulation of cyclic AMP and increased phosphodiesterase activity; adenylate cyclase activity was depressed. It is suggested that dopa exerts its effect not only as a precursor of melanin, but also through the cyclic AMP system, affecting the activity of enzymes of melanogenesis.Deceased.Department of Biochemistry, S. M. Kirov Military Medical Academy, Leningrad. V. G. Khlopin Radium Institute, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR A. N. Klimov). Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 4, pp. 425–426, April, 1978.     

Taenia solium cysticerci synthesize androgens and estrogens in vitro

Cysticerci from Taenia solium develop in the pig muscle and cause severe diseases in humans. Here we report on the capacity of T. solium cysticerci to synthesize sex steroid hormones. T. solium cysticerci were dissected from infected pork meat. Parasites were incubated for different periods in culture media plus antibiotics and tritiated steroid precursors. Blanks and parasite culture media were extracted and analyzed by thin-layer chromatography (TLC) in two different solvent systems. In some experiments, the scoleces were incubated separately. Results showed that T. solium cysticerci transform [³H]androstenedione to [³H]testosterone in a time-dependent manner. The production was confirmed in two different solvent systems. The incubation with [³H]testosterone yielded only small amounts of [³H]androstenedione. The recrystallization procedure further demonstrated that the metabolite identified by TLC was testosterone. The isolated scoleces incubated in the presence of [³H]androstenedione yielded [³H]testosterone and small quantities of [³H]17β-estradiol. The results reported here demonstrate that T. solium cysticerci have the capacity to synthesize steroid hormones.     

Autoradiographic localization of [3H]reserpine in rat brain: Correlation with distribution of monoaminergic neurones

The in vivo localization of [³H]reserpine in rat brain was studied by autoradiography using unfixed, frozen sections prepared 18 h after intravenous injection of the radiolabel. Radioactivity was localized in those areas of the brain where monoaminergic cell bodies and nerve terminals have been describes. The radiolabel found was chromatographically identical with reserpine. Treatment of animals with the unlabelled drug 4 h before administration of [³H]reserpine prevented the specific localization of radioactivity in the brain.     

Mapping monoaminergic neurons with [3H]reserpine by autoradiography.

The synthesis of [³H]reserpine of high specific activity is described. The accumulation of radioactivity in peripheral sympathetically innervated organs and in the brain after intravenous injection of [³H]reserpine to rats was measured biochemically and its localization studied by light-microscopic autoradiography. In most of the organs and tissues investigated minute quantities of [³H]reserpine persisted up to 10 days after injection. By autoradiography, it was observed that silver grains were unevenly distributed in various brain regions and peripheral organs 18 h and up to 10 days after administration of [³H]reserpine. In the brain the radiolabel selectively accumulated in distinct areas known to have a high monoamine content including the substantia nigra, nucleus (n.) raphe, locus coeruleus, n. dorsomedialis hypothalami and the n. arcuatus, where cell bodies as well as the neuropil were labelled; whereas in the neostriatum, n. septi lateralis, n. accumbens, n. tractus solitarii, eminentia mediana, hippocampus and cortex the label was confined to the neuropil. Supra-ependymal 5-hydroxytryptamine-containing nerves in the cerebroventricular system and the interplexiform layer of the retina also selectively accumulated the label, as did a network of nerve fibres in the iris, cells and paracellular areas of the superior cervical ganglia and chromaffin cells of the adrenal medulla. Pretreating (but not post-treating) animals with non-radioactive reserpine prevents, by up to 80%, the accumulation of radiolabel and abolishes to a great extent the autoradiographic localization. The fact that the persistently bound radiolabel is confined to monoaminergic neurons suggests that it is irreversibly bound to its target site, the amine-storing vesicle. Support for this interpretation comes from studies demonstrating a fast anterograde axonal transport of [³H]reserpine in the nigrostriatal tract after intranigral injection of the radiolabel.Altogether, these findings are in line with a wealth of neuropharmacological, endocrinological and clinical observations relating the effects of reserpine to its interaction with dopaminergic, noradrenergic and serotonergic neuronal systems. They demonstrate that [³H]reserpine of high specific activity is a useful tool in studies designed to map monoaminergic pathways in the brain and to further characterize amine-storing mechanisms.     

Polyacrylamide gel electrophoresis of the RNA products labeled in vitro by extracts of leaves infected with bromegrass mosaic virus

The RNA products synthesized in vitro by a crude RNA polymerase preparation from barley leaves infected with bromegrass mosaic virus (BMV) were analyzed by electrophoresis in polyacrylamide gels. The following labeled products were identified depending upon the incubation time with [³H]UTP: (1) slow-migrating products whose radioactivity was removed by treatment with RNase; (2) double-stranded replicative forms, and (3) single-stranded RNAs which migrated at the positions of the major genomic BMV-RNAs. This labeling was specific for preparations made from BMV-infected leaves and required the presence of all 4 ribonucleotides.A limited amount of radioactivity was chased into the various components of BMV-RNA from the slow-migrating pulse-labeled product, most of which did not turn over rapidly under the chase conditions used.     

Binding of [3H]ICIA 5165, an H2-receptor antagonist to guinea pig gastric mucosa

ICIA 5165, 2-guanidino-4-[4-(2-cyano-3-methylguanidino)butyl] thiazole, a selective histamine H₂-receptor antagonist was radiolabelled with tritium to a specific activity of 50.8 Ci/mmoll for use in binding studies. Radiolabelling did not impair bioactivity.Binding characteristics of [³H]ICIA 5165 to guinea pig gastric mucosa were determined. Ligand binding was rapid, reaching equilibrium within five minutes at 0°C, reversible and saturable. Specific [³H]ICIA 5165 binding had an equilibrium dissociation constant of 1.29×10^–8 M, determined by Scatchard plot analysis, and of 1.02×10^–8 M, calculated from the ratio of the dissociation to association rate constants. A Hill number, n_H, of 1.02 was determined for the specific binding component. Specific binding of [³H]ICIA 5165 to gastric mucosal supernatant was not inhibited by methapyrilene, diphenhydramine, mepyramine, d-chlorpheniramine or I-chlorpheniramine (all at 10^–7 M), or by atropine or propranolol (both at 10^–6 M). Specific [³H]ICIA 5165 binding was inhibited in a concentration dependent manner by non-radioactive ICIA 5165 and tiotidine, as well as by a variety of other agents, with H₂ agonist or H₂ antagonist properties. In competition experiments, however, difficulties encountered in accurately defining the degree of specific binding indicate some reservation should be observed in interpreting these results.