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

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

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.     

Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores

P hillips , A.P. & M artin , K.L. 1984. Radioactive labels for Protein A: evaluation in the indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores. Journal of Applied Bacteriology 56 , 449–456.
Staphylococcus aureus Protein A (SpA) labelled with [¹²⁵I] by the Bolton & Hunter (1973) method performed about as well as labelled sheep anti-rabbit globulin (SAR) in an indirect immunoradiometric assay (IRMA) for Bacillus anthracis spores immobilized on multispot microscope slides. SpA labelled with [³H] by propi-onylation also performed well but would be expensive to use. SpA labelled with [³H] fluorodinitrobenzene, or labelled with [¹²⁵I] by the chloramine T reaction gave erratic assay results, high noise values and low signal-to-noise ratios, indicating substantial direct binding of labelled SpA to the slide surface and to the bacterial preparation. The uptake of radioactively labelled SpA in the IRMA was compared with the fluorescence intensity of individual spores in a micro-fluorometric immunofluorescence (IF) test involving dual labelled fluorescein-[¹²⁵I]-SpA. The maximum number of SAR molecules bound to the mixture of spores and cell-free antigens in the B. anthracis IRMA was about twice the maximum number of radioactively labelled SpA molecules bound. The SAR : SpA saturation binding ratio on the surface of the spores, however, was approximately the inverse of this. It is concluded that radioactively-labelled SpA is not recommended in preference to anti-species antibody reagents in bacterial IRMA tests but fiuorescein-conjugated SpA deserves further consideration for use in microscope-based IF tests for bacterial antigens.     

Binding of type-I and type-II collagens to Staphylococcus aureus strains isolated from patients with toxic shock syndrome compared to other staphylococcal infections

Abstract Toxic shock syndrome toxin-1 (TSST-1) producing strains of Staphylococcus aureus isolated from 18 patients with toxic shock syndrome (TSS) and from 56 patients with other diagnoses were compared for capacity to interact with various serum and connective tissue proteins. TSS associated isolates showed significantly stronger binding of Type-I collagen (Cn-I) and Cn-II than non-TSS strains, in a particle agglutination assay (PAA) as well as in ¹²⁵I labelled Cn uptake experiments. ¹²⁵I Cn-IV binding, was similar between the two groups, whereas in PAA, a stronger interaction was observed for non-TSS than TSS associated strains. The median binding of ¹²⁵I Cn to TSS-associated strains were 52.2 (Cn-I), 30.6 (Cn-II) and 20.0 (Cn-IV) compared to 20.0 (Cn-I), 14.4 (Cn-II) and 24.4 (Cn-IV) values of non-TSS strains. A saturation with ¹²⁵I Cn-I and Cn-II binding was established for TSS (30 min) and non-TSS (15 min) strains. ¹²⁵I Cn-IV binding reached a saturation in 10 min and 90 min with TSS and non-TSS strains respectively. Finally, the binding profiles of TSS associated and non-TSS strains to fibronectin, fibrinogen, laminin and IgG did not differ in both PAA and radioisotope assays. In scanning electron microscopy, cells of TSS associated strains bound to the reprecipitated native Cn-I fibrils. In contrast, most cells of non-TSS strains were localized to the distal end or were trapped between the Cn fibrils. The stronger interaction with Cn-I and II in particular, shown by TSS associated strains, might enhance submucosal localization, thereby facilitating entry of toxins into the blood and establishment of TSS.     

Radioiodination of Enterotoxin from Clostridium perfringens Type A using Chloramine T

Procedures were examined for labelling enterotoxin isolated from Clostridium perfringens type A. with ¹²⁵I using chloramine T as the oxidizing agent. The iodination method was evaluated critically to establish the optimal conditions for the preparation of iodinated enterotoxin with a high specific radioactivity and without impairing the immunospecificity and biological activity. The use of 250 μg/ml of chloramine T in the reaction mixture. 500–1000 μCi of Na¹²⁵I/10 μg of enterotoxin and a reaction time of 40 s at pH 7–0 produced ¹²⁵I-enterotoxin of both high specific radioactivity and immunospecificity which retained its biological activity. No damage or aggregate formation due to the iodination process was observed. Enterotoxin labelled with high specific activity (135 μCi μg) showed extensive dissociation of ¹²⁵I when stored at 4°C and—20°C. In contrast, toxin labelled with low specific activity (7 μgCi/μg) was stable for as long as two months. The immunoreactivity of all labelled preparations was essentially unchanged after storage for one month.     

Cerebral clearance of human amyloid-beta peptide (1-40) across the blood-brain barrier is reduced by self-aggregation and formation of low-density lipoprotein receptor-related protein-1 ligand complexes

Soluble amyloid-β peptide (Aβ) exists in the form of monomers and oligomers, and as complexes with Aβ-binding molecules, such as low-density lipoprotein receptor-related protein-1 (LRP-1) ligands. The present study investigated the effect of self-aggregation and LRP-1 ligands on the elimination of human Aβ(1–40) [hAβ(1–40)] from the rat brain across the blood–brain barrier. Incubation of [¹²⁵I]hAβ(1–40) monomer resulted in time-dependent and temperature-dependent dimer formation, and the apparent elimination rate of [¹²⁵I]hAβ(1–40) dimer was significantly decreased by 92.7% compared with that of [¹²⁵I]hAβ(1–40) monomer. Pre-incubation with LRP-1 ligands, such as activated α2-macroglobulin (α2M), apolipoprotein E2 (apoE2), apoE3, apoE4, and lactoferrin, reduced the elimination of [¹²⁵I]hAβ(1–40). By contrast, pre-administration of the same concentration of these molecules in the rat brain did not significantly inhibit [¹²⁵I]hAβ(1–40) monomer elimination. Purified [¹²⁵I]hAβ(1–40)/activated α2M complex and [¹²⁵I]activated α2M were not significantly eliminated from the rat brain up to 60 min. MEF-1 cells, which have LRP-1-mediated endocytosis, exhibited uptake of [¹²⁵I]activated α2M, and enhancement of [¹²⁵I]hAβ(1–40) uptake upon pre-incubation with apoE, suggesting that [¹²⁵I]activated α2M and [¹²⁵I]hAβ(1–40)/apoE complex function as LRP-1 ligands. These findings indicate that dimerization and LRP-1-ligand complex formation prevent the elimination of hAβ(1–40) from the brain across the blood–brain barrier.     

Effect of Vinblastine On the Cell Cycle and Migration of Ameloblasts of Mouse Incisors As Shown By Autoradiography Using 3H-Thymidine

Abstract. The effects of vinblastine on the cell cycle and the migration of ameloblasts were studied in the lower incisors of mice by labelling the cells with ³H-thymidine ([³H]TdR) and radioautography. A group of mice received 2 μg/g of body weight vinblastine intraperitoneally and 6 hr after these animals and those of a control group were injected with 1μCi/g body weight of [³H]TdR, and sacrificed at time intervals from 0.75 hr to 15 days.
The generation time of ameloblasts in the progenitor compartment was 14.8 hr in animals treated with vinblastine and 17 hr in the controls, using the FLM curve method; with the grain dilution method the duration was respectively 29.25 hr and 25.96 hr. the thymidine labelling index of the treated animals was 50% higher than the controls. the velocity of ameloblast migration, determined either by the displacement of the most incisally labelled cell or by the grain dilution method, was lower in the experimental group (2.48 cell positions/hr and 9.18 μ/hr respectively) as compared with the control (3.21 cell positions/hr and 18.88 μm/hr respectively).
The results on the ameloblast production rate are contradictory but the slowing down in the velocity of cell migration is compatible with a decrease of the rate of cell production in the progenitor compartment as a vinblastine effect.     

[1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES

Abstract— [¹²⁵I]Diiodo α-bungarotoxin ([¹²⁵I]₂BuTx) and [³H]quinuclidinylbenzilate ([³H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O -acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.
All species and regions except Drosophila had 16–150 fold more [³H]QNB binding sites than [¹²⁵I]₂BuTx binding sites. Brain regions with the highest levels of [¹²⁵I]₂BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [³H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [³H]QNB and [¹²⁵I]₂BuTx binding were seen in cerebellum. The utility of [¹²⁵I]₂BuTx and [³H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.     

Local Re-Utilization of Thymidine In Normal Mouse Tissues As Measured With Iododeoxyuridine

Abstract. Thymidine (TdR) and its analogue, iododeoxyuridine (IdUdr), were used to quantitate nucleoside re-utilisation in vivo. Significantly different results are obtained, however, depending upon what form of isotopically labelled iododeoxyuridine is used. No measurable local thymidine re-utilization was found in mouse thymus, spleen or bone marrow when the retention of [³H]IdUdR was compared with [¹⁴C]TdR. On the other hand, significant differences were found between the retention of [¹²⁵I]IdUdR and [³H]IdUdR, which is attributed to de-iodination of iododeoxyuridine. Some thymidine re-utilization was found in duodenum using both [³H]IdUdR and [¹²⁵I]IdUdR. Information on the in vivo distribution of TdR and the contention that a large degree of TdR re-utilization in the thymus is evidence of extensive cell death must be re-interpreted in the light of these results. In addition, evidence for little or no local re-utilization in some tissues will greatly simplify the use of [¹¹C]TdR as an imaging agent for measuring tissue proliferation in vivo with positron emission tomography (PET).     

Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT_1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT_1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin- O -carboxymethylglycyl[¹²⁵I]iodotyrosinamide ([¹²⁵I]GTI; a selective 5-HT_1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT_1B receptors. The remaining 40% of [¹²⁵I]GTI binding sites were shown to be 5-HT_1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT_1B/1Dβ, but not the 5-HT_1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT_1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [¹²⁵I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT_1Dβ component of [¹²⁵I]GTI specific receptor binding sites.     

Methyl 3β-(4-[125I]Iodophenyl)Tropane-2β-Carboxylate In Vitro Binding to Dopamine and Serotonin Transporters Under "Physiological" Conditions

Abstract: Methyl 3β-(4-[¹²⁵I]iodophenyl)tropane-2β-carboxylate ([¹²³I]β-CIT) is a single photon emission computed tomographic radiotracer for in vivo labeling of dopamine (DA) and serotonin (5-HT) transporters. Single photon emission computed tomographic experiments in nonhuman primates showed that [¹²³I]β-CIT in vivo binding to DA transporters had a much slower washout than binding to 5-HT transporters. This observation was not predicted from previously published in vitro studies. These studies, performed at 22°C in nonphysiological buffer, reported similar affinity of [¹²⁵I]β-CIT for DA and 5-HT transporters. We now report [¹²⁵I]β-CIT binding parameters to fresh rat membranes at 22°C and 37°C, in a buffer mimicking the composition of cerebrospinal fluid. At both temperatures, binding to DA transporters was best fit by a twosite model, whereas binding to 5-HT transporters was compatible with one population of sites. At 22°C, [¹²⁵I]β-CIT showed similar affinity to high-affinity DA (0.39 n M ) and 5-HT transporter sites (0.47 n M ). Increasing the incubation temperature from 22°C to 37°C reduced binding to DA transporters by 60%, whereas binding to 5-HT transporters was only marginally affected. In vitro kinetic experiments failed to detect significant differences in on or off rates that could explain the observed in vivo kinetics. These experiments thus failed to explain [¹²³ I]β-CIT in vivo uptake kinetics, suggesting the existence of specific factors affecting the in vivo situation.     

5-Methoxytryptamine Inhibits Cyclic AMP Accumulation in Cultured Retinal Neurons Through Activation of a Pertussis Toxin-Sensitive Site Distinct from the 2-[125I]Iodomelatonin Binding Site

Abstract: Melatonin and 5-methoxytryptamine inhibited forskolin-stimulated cyclic AMP formation in cultured neural cells prepared from embryonic chick retina. Both methoxyindoles exhibited similar potency and efficacy, with EC₅₀ values of 0.8 n M for melatonin and 7.2 n M for 5-methoxytryptamine. Inhibition of cyclic AMP formation by 5-methoxytryptamine or melatonin was prevented by pretreatment with pertussis toxin. Pretreatment of cultures with 5-methoxytryptamine for 24 h reduced the subsequent inhibitory cyclic AMP response to 5-methoxytryptamine but not that to 2-iodomelatonin. Putative melatonin receptors on cultured retinal cells were labeled with 2-[¹²⁵I]iodomelatonin. Melatonin displaced specific 2-[¹²⁵I]iodomelatonin with a K _i value (0.8 n M ) similar to the EC₅₀ for inhibition of cyclic AMP formation. In contrast, 5-methoxytryptamine only inhibited 2-[¹²⁵I]iodomelatonin binding at very high concentrations ( K _i = 650 n M ). Pretreating cultured cells for 24 h with 2-iodomelatonin or melatonin, but not with 5-methoxytryptamine, reduced subsequent 2-[¹²⁵I]iodomelatonin binding. Thus, 5-methoxytryptamine appears to inhibit forskolin-stimulated cyclic AMP formation at a site distinct from the 2-iodomelatonin binding site.     

LACTOPEROXIDASE-COUPLED IODINATION OF BOVINE CHROMAFFIN GRANULES

Abstract— Chromaffin granules were iodinated with lactoperoxidase at either their external or internal membrane surfaces. When iodination of internal soluble granule proteins and membrane phospholipids was minimized, the majority of the membrane proteins, including the 83,000 component, were iodinated. Components with molecular weights 63,000, 61,000, 51,000, 44,000, 32,000, 26,000 and 19,000 had a higher ¹²⁵I specific activity than did the other membrane components, suggesting they were more accessible at the outer membrane surface than were the other components. In the presence of detergent, the iodination of all membrane components was increased more than 10-fold; the incorporation of ¹²⁵I was now similar to their Coomassie Blue staining intensity in disc gels, indicating that all components were equally accessible to lactoperoxidase. In the presence of detergent, iodine incorporation into the MW 83,000 and 16,000 components was stimulated approx 100-fold.
The MW 83,000, 63,000, 61,000 and 37,000 components incorporated significant amounts of ¹²⁵I when granule membranes were iodinated from their internal surface, suggesting these components have a portion of their polypeptide chain accessible at the inner membrane surface. Thus the MW 83,000 component, which we identified as dopamine β hydroxylase, and the MW 63,000/61,000 components, which are part of the membrane ATPase, can be iodinated from both membrane surfaces. This would suggest that these are transmembrane proteins. However, the major portion of all the proteins in this membrane were inaccessible to lactoperoxidase at either membrane surface.     

Heterogeneity of β-Adrenoceptors in Guinea-Pig Brain: Radioligand Binding and Cyclic Nucleotide Generation

Abstract: In this report, we have examined the radioligand binding and second messenger signalling characteristics of β-adrenoceptors in the guinea-pig brain. [¹²⁵I]lodocyanopindolol ([¹²⁵I]ICYP)-labelled sites in the cerebellum and cerebral cortex were of similar densities ( B _max 34 and 24 fmol·mg⁻¹) and affinities ( K _D 20 and 55 p M ), respectively. Analysis of competition for [¹²⁵I]ICYP binding in the cerebellum was compatible with the presence of a β₂-adrenoceptor. In this tissue, isoprenaline evoked a cyclic AMP stimulation, and also potentiated cyclic GMP accumulations evoked in the presence of a nitric oxide donor, consistent with mediation via a β₂-adrenoceptor. The [¹²⁵I]ICYP binding profile in the cerebral cortex did not comply with those previously described for β-adrenoceptor subtypes, and isoprenaline failed to alter significantly cyclic AMP accumulation in the cerebral cortex, hippocampus, or neostriatum, even in the presence of forskolin or a phosphodiesterase inhibitor. Isoprenaline was also without effect on cyclic GMP accumulation or phosphoinositide turnover in the cerebral cortex. These results suggest that the guinea-pig cerebellum expresses a functional β₂-adrenoceptor coupled to cyclic AMP generation, and potentiation of cyclic GMP accumulation. However, the guinea-pig cerebral cortex displays binding sites that exhibit β-adrenoceptor-like pharmacology but fail to show functional coupling to cyclic AMP, cyclic GMP, or phosphoinositide signalling systems.     

DNA SYNTHESIS TIME IN LEUKAEMIC CELLS AS MEASURED BY THE DOUBLE LABELLING AND THE PERCENTAGE LABELLED MITOSES METHODS

Values of T _s provided by the double labelling method have been compared with those given by the percentage labelled mitoses curve for blast cells in the peripheral blood of a patient with plasma cell leukaemia and of rats bearing a transferable acute leukaemia. the double labelling method was carried out giving the first label (³H-thymidine) in vivo and the second label (¹⁴C-thymidine) in vitro with several values for the interval between the two labels. T _s was calculated by fitting regression lines to the results obtained. Data for percentage labelled mitoses were analysed by computer. For the plasma cell leukaemia values of T _s= 17.1 ± 7.0 hr and T _s= 19.8 ± 3.4 hr, and for the rat leukaemia values of 8.7 ± 1.7 hr and 9.0 ± 1.7 hr (7.1 hr corrected for exponential growth) were obtained from the percentage labelled mitoses and double labelling methods respectively. It is concluded that the double labelling method is valid for the study of cell proliferation in leukaemic blast cells.     

An Indirect Radiolabelled Antibody Staining Technique for the Rapid Detection and Identification of Bacteria

Small numbers of Bacillus subtilis var. niger spores, Serratia marcescens and Francisella tularensis can be detected and identified in an hour using an indirect radiolabelled antibody staining technique with homologous rabbit antibacterial antibody and radiolabelled goat anti-rabbit globulin. Tritium is better than ¹²⁵I, ¹⁴C and ³⁵S for labelling the globulin. The reagent obtained is sensitive and adequately stable.     

Normal or Retrovirus-Infected Cultured Chicken Embryo Cells Express the 48,000 D Age-Related Antigen Characteristic of Embryonic Chicken Erythrocytes

The surface of normal or retrovirus-infected chick embryo cells was labelled with ¹²⁵I using lactoperoxidase. The solubilized membrane material was allowed to react with antisera raised in rabbits to cultured chick embryo cells or to the membranes of embryonic or adult chicken erythrocytes. Analysis of the immunoprecipitates shows that chicken embryo cells not of erythropoietic origin express on their surface membrane an antigenic polypeptide of mol. wt. 48,000 daltons (D), which appears to be identical with that expressed on embryonic chicken erythrocytes.     

Development of Polyclonal Anti-D2 Dopamine Receptor Antibodies to Fusion Proteins: Inhibition of D2 Receptor–G Protein Interaction

Abstract: Portions of the cDNA encoding the third intracellular loop (i3 loop) of the long and short isoforms of the rat D2 dopamine receptor were subcloned into the vector pNMHUBpoly and expressed in Escherichia coli as fusion proteins. The fusion proteins were gel-purified and used to immunize rabbits for the production of polyclonal anti-receptor antisera. The anti-fusion protein antisera recognized synthetic peptides corresponding to segments of the i3 loops of D2 dopamine receptors in a solid-phase radioimmunoassay. Antisera were tested in an immunoprecipitation assay using the reversible D2 antagonist [¹²⁵I]NCQ 298 and digitonin-solubilized extracts of canine and rat caudate. [¹²⁵I]-NCQ 298 bound reversibly and with high affinity (K_D= 0.14 n M ) to receptors in solubilized extracts enriched by chromatography on heparin-agarose. The anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to immunoprecipitate quantitatively D2 dopamine receptors labeled with [¹²⁵I]NCQ 298 from solubilized rat caudate. The antibodies were tested for their ability to affect the coupling of D2 dopamine receptors to GTP-binding proteins in digitonin-solubilized rat caudate. Both anti-UBI-D2i3_L and anti-UBI-D2i3_s antisera were able to inhibit the high-affinity binding of the agonist N -propylnorapomorphine to digitonin-solubilized rat caudate. These findings indicate that the i3 loop of the D2 dopamine receptor is an important determinant for coupling of the G protein.     

AUTORADIOGRAPHIC INVESTIGATION ON THE CELL KINETICS OF CRYPT EPITHELIA IN THE JEJUNUM OF THE MOUSE °CONFIRMATION OF STEADY STATE GROWTH WITH CONSTANT FREQUENCY DISTRIBUTION OF CELLS THROUGHOUT THE CYCLE

Cell kinetic parameters of cells in the crypt of the jejunum of the mouse were obtained autoradiographically. A number of different methods used in cell proliferation studies were applied to the same animal strain kept under constant conditions. In order to avoid effects of geometrical factors, squashes of isolated crypts were used.
The generation time was determined by the per cent labelled mitoses method of Quastler, modified by double labelling with ³H- and ¹⁴C-TdR. This modified method permits a more exact determination of the generation time. The duration of the cycle was 14 hr.
Double labelling experiments in which an injection of ³H-TdR was followed by an injection of ¹⁴C-TdR after 1 hr showed that the cell flux was 7.0%/hr at the beginning of the S-phase and 7.68%/hr at the end. Assuming steady state growth a constant cell flux of 7.15%/hr within the whole cycle can be derived from the measured generation time of 14 hr. These results clearly show that the crypt epithelia constitute a steady state system with constant frequency distribution of the cells throughout the cycle.
The per cent labelled mitoses method after a single injection of ³H-TdR as well as double labelling experiments with ³H- and ¹⁴C-TdR give an estimate of the S-phase of 8.0 or 7.4 hr respectively. Double determinations lead to a value of 0.54 or 0.52 hr respectively for the duration of mitosis and to values of 77% and 72%     

Translocation of amino acids from stem nodules of Sesbania rostrata demonstrated by GC-MS in planta 15N isotope dilution

We report a novel use of the ¹⁵N dilution technique to detail the translocation of amino compounds in the legume Sesbania rostrata . The conventional ¹⁵N dilution technique follows the dilution of ¹⁵N within a labelled plant, as ¹⁴N₂ is fixed by symbiotic bacteria. In our experiments, stem-nodulated Sesbania rostrata were enriched by feeding with ¹⁵N ammonium nitrate for 2 weeks, followed by a 1 week period where the only N available to the plants was via nitrogen fixation of atmospheric N₂. We measured the composition, concentration and ¹⁵N enrichment of amino compounds in various plant tissues, both above and below the stem nodules, using GC-MS and isotopic abundance mass spectrometry techniques. Approximately 28% of the total N in the stem nodules was derived from internal plant sources. The ureides allantoic acid and allantoin were not abundant in xylem, leaf or nodule tissues. The amides asparagine and glutamine were the major export products from stem nodules although a wide range of other amino compounds are also synthesized. Amino acids within the nodules had a low level of enrichment, demonstrating that a small fraction (≈ 11%) was derived from outside the nodules, and significant cycling of N (28% of xylem N) through the root system was revealed by measurements of ¹⁵N distribution and amino acid concentrations.