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PubMed1986

Uptake and metabolism of indole compounds by the goldfish pineal organ.

Indole metabolism was studied in the pineal organ of the goldfish by radioautography and high-performance liquid chromatography. The rate of uptake of tritiated serotonin was rapid in vitro with dense labeling over the photoreceptor cells. Tritiated tryptophan was taken up at a slower rate and the label was distributed evenly over the epithelium. Continual light caused a reduction in the concentration of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) compared to groups exposed to constant darkness both in vivo and in explants, suggesting that these effects are not derived from photoreceptors outside the pineal organ. These data are consistent with the hypothesis that indole metabolism is functionally linked to phototransduction events in the pineal organ of lower vertebrates.

Animals↗

PubMedDate not supplied

Cell proliferation in the atherosclerotic plaques of cholesterol-fed rabbits. Part 3. Histological and radioautographic observations on glucocorticoids-treated rabbits.

Tritiated thymidine radioautography was employed to study the effect of cortisol and other glucocorticoids on cellular proliferation in the aorta and pulmonary artery of rabbits with cholesterol atherosclerosis. Labelled cell counts showed that glucocorticoids, even after one day and at a relatively low dose, decrease sharply the deoxyribonucleic acid synthesis in the intimal plaques. The hormonal influence on [3H]thymidine uptake seems to be a dose-dependent process. The relative potency of these steroids in inhibiting DNA synthesis in the plaques parallels closely their anti-inflammatory effectiveness. Conversely mineralocorticoids, including aldosterone and deoxycorticosterone, increase the rate of DNA synthesis in the plaques. It is concluded that the antiatherogenic effect of glucocorticoids on cholesterol-fed rabbits may be due, at least partly, to the inhibitory effect of these steroids on the DNA synthesis of the cellular components of the intimal plaques.

Animals↗

PubMed1979

The B lymphocyte-deprived mouse as a tool in immunobiology.

The purpose of this article is to demonstrate that mice subjected to continuous injections with anti-IgM antibodies, initiated within one day of birth, or in adulthood, lose all their B lymphocyte functions, but retain T lymphocyte reactivity. The B lymphocyte status of these mice was analyzed by histology, radioautography, serum immunoglobulin levels, the antibody response and reactivity to LPS in vitro. T lymphocyte functions were evaluated in vivo by tumor allograft rejection, contact sensitization, and in vitro by reactivity to mitogens and allogeneic cells. On the basis of the results described, it is suggested that the anti-IgM-treated mouse may be considered as a balid and useful experimental model of the "T mouse".

Aging↗

PubMed1980

111Indium-labeled human alveolar macrophages and monocytes: function and ultrastructure.

Human alveolar macrophages and peripheral blood mononuclear cells were labelled with indium-111 (111In)-oxine in ethanol. The efficiency of labeling averaged 84% for macrophages and 74% for mononuclear cell preparations. Phagocytosis by macrophages, the release of colony stimulating factor activity (CSF) by macrophages, and chemotaxis by monocytes in response to human C5-derived chemotactic factor (C5fr) and formyl-methionyl-leucyl-phenylalanine (f-MLP), were indistinguishable between labeled and unlabeled cells. Microscopically, labeled cells looked similar to control cells. Radioautography demonstrated 111In throughout the labeled cells, but there was a disproportionately high quantity over nuclei. These findings add to the growing literature indicating that 111In labels cells efficiently and that cells can retain function after being labeled with 111In.

Autoradiography↗

PubMed1980

Sandwich radioimmunolabeling for the study of surface properties of bone marrow lymphocytes.

A modification of sandwich radioautographic method was applied to the study of surface immunoglobulin and/or specific antigens on small lymphocytes in mouse and human bone marrow. After incubation of marrow cell suspensions at 37 degrees C, cells were reacted at 0 degree C for 30 min with graded dilutions of rabbit anti-mouse or anti-human immunoglobulin followed by further reaction with a sheep anti-rabbit immunoglobulin labeled with 125I. Detectable surface immunoglobulin was demonstrated in approximately one-third of mouse marrow lymphocytes and 20-25% of human marrow lymphocytes. The densities of surface immunoglobulin as assessed by grain counts on individual labeled lymphocytes tended to be lower in the marrow than in spleen or peripheral blood. When the same rabbit antiserum was used to compared the sensitivity of the sandwich method with that of the direct radioautography, the former was found sufficiently sensitive to give a plateau level of labeling without seriously increasing background grains. The advantages of the method are discussed with reference to studies on T and B cells specific antigens on human bone marrow lymphocytes.

Animals↗

PubMed1981

Sensitive measurement of endotoxin by radio-rocket immunoelectrophoresis using [125I] Staphylococcus aureus protein A.

Antibody directed against the core glycolipid antigen (CGL) of the mutant Salmonella minnesota Re 595 has been shown to cross-react with endotoxin from bacteria within the group Enterobacteriaceae. Using this cross-reactive CGL antibody we have developed a sensitive (250 pg) radio-rocket immunoelectrophoretic technique to measure endotoxin. We used the principles of rocket immunoelectrophoresis and increased the sensitivity by using 125 I-labelled staphylococcal protein A which serves as a sensitive probe to bind to the Fc portion of the IgG complexed with antigen. The rocket-shaped [125I] protein A labelled immune complexes were detected by radioautography. The sensitivity is 100-fold greater than conventional Coomassie brilliant blue staining. Measurement of CGL was inhibited by normal human serum. However, the assay had the capacity to quantitate endotoxin in buffer extracts of clinically isolated Escherichia coli, Serratia marcescens, Klebsiella pneumoniae but not Pseudomonas aeruginosa. Analysis of various preparations of CGL obtained from different investigators demonstrated wide variation in their immunoreactivity. Because of the significant cross-reaction to detect various endotoxins this method has the potential to measure endotoxemia and assess the immunochemical quality of various endotoxin preparations. Additionally, the technique of using [125I] protein A has wide applicability for the sensitive measurement of other antigens.

Antigens, Bacterial↗

PubMed1984

The localization of B lymphocytes in mouse bone marrow: radioautographic studies after in vivo perfusion of radiolabelled anti-IgM antibody.

An in vivo cell surface labelling technique using radioautography has been developed to visualise the distribution of IgM-bearing B lymphocytes within the bone marrow. Anaesthetized 3-week-old mice were perfused via the common iliac artery with: (1) serum-containing medium (SCM), (2) 125I-labelled anti-IgM antibody in SCM, (3) SCM, and (4) fixative. In radioautographic sections of femoral marrow labelled surface IgM+ cells were observed either singly or in small clusters throughout the extravascular haemopoietic marrow cords. Binding specificity was demonstrated by the displacement of 125I-anti-IgM labelling by excess anti-IgM and by the binding of perfused 125I-anti-H-2Kk antibody in CBA/J (H-2Kk) mice but not in C57BL/6 (H-2Kb) mice. Quantitative analysis of radioautographic sections revealed an even distribution of labelled cells throughout CBA/J marrow perfused with 125I-anti-H-2Kk, indicating a uniform accessibility of perfused antibody to cells in the haemopoietic cords. This labelling pattern contrasted with that in 125I-anti-IgM perfused animals in which surface IgM+ cells, although widely distributed in the bone marrow, showed areas of concentration, speculatively clones of maturing B lymphocytes. This method of labelling surface IgM and other cell markers in situ provides an approach to study the microenvironment of B lymphocyte genesis in the bone marrow.

Animals↗

PubMed1984

A simple general method of exactly comparing different binding activities and antigenic properties of antibodies by obtaining duplicate copies from a single isoelectric focusing gel.

We report a simple general method of exactly comparing different binding activities and/or antigenic properties of antibodies in ascites fluid after fractionation on a single isoelectric focusing gel. Two copies of the gel are made on nitrocellulose: 1 copy is overlaid with the radioiodinated form of one overlay protein and the other copy is overlaid with the radioiodinated form of another. By superimposing X-ray films obtained after radioautography, any 2 binding activities or antigenic properties of antibodies can be compared exactly and in detail. The procedure is simple, highly sensitive, and independent of the molecular weight of the overlay protein. This is accomplished without requiring isolation of the antibodies, denaturing agents, precipitation, chemical fixation or other modification reactions.

Acrylic Resins↗

PubMed1985

Affinity labeling of the Fc receptor on human monocytes using bifunctional cross-linking agents.

To affinity label the Fc receptor on human monocytes, Fc fragments of monoclonal human IgG1 radiolabeled with iodine 125 were covalently bound to the surface of intact monocytes using a variety of bifunctional cross-linking agents including ethylene glycol bis(succinimidyl succinate), dithio-bis-(succinimidyl proprionate), maleimidobenzoyl N-hydroxysuccinimide, glutaraldehyde and dimethyl suberimidate. After cross-linking, cells were solubilized and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by radioautography. Each of these cross-linkers caused a portion of cell-bound Fc fragments to form a covalent complex with a monocyte membrane component. This complex migrated on electrophoresis with an apparent molecular weight of 120,000. Deducting the molecular weight of Fc fragments alone (53,000) the molecular weight of the second component of the complex therefore was about 67,000. A similar estimate of receptor size also was obtained after reduction with dithiothreitol. Complex formation was potently inhibited by unlabeled Fc fragments, IgG1 or IgG3, all of which would be expected to compete with Fc fragments for IgG Fc receptor on human monocytes, but was not inhibited by Fab fragments, IgG2 or IgG4, which do not bind avidly to this receptor. By quantitating the amount of complex formed in the presence of varying concentrations of labeled ligand, it could be demonstrated that complex formation was saturable, and that Fc fragments formed complexes with avidity comparable to that with which Fc fragments bound to receptors on intact monocytes. The findings establish the feasibility of using radiolabeled Fc fragments to affinity label the IgG Fc receptors on human leukocytes. Potential advantages of this approach to studying receptor structure are discussed.

Affinity Labels↗

PubMed1986

Preparation and properties of antibodies doubly labeled with tritium and fluorescein.

A method was developed to prepare doubly labeled antibodies whereby fluorescein-labeled antibodies were reacted with N-[ethyl-2-3H]ethylmaleimide. This tritiated conjugate exhibited the same immunoreactivity as the non-derivatized fluorescein-labeled antibody. This method provides a marker for immunocytochemical studies which takes advantage of the rapidity of fluorescent tracing combined with the stability and precision of the radioautography technique.

Animals↗

PubMed1992

A cleavable biotinylating agent and its use in protein electroblots.

A direct method for the synthesis of N-biotinyl penicillamine is described. It has been shown to be a convenient biotinylating agent for antibodies which have been previously coupled with SPDP. The biotinylated antibodies can be used to detect antigens on protein electroblots using 125I-labelled streptavidin and radioautography. The biotin and its attached streptavidin and radiolabel can be removed under mild conditions and the blot reprobed with a different antibody using an identical protocol.

Antibodies, Monoclonal↗

PubMed1985

Metabolism of progesterone by healthy and inflamed human gingiva in vitro.

[4-14C]Progesterone was incubated with homogenate and mitochondrial, microsomal and soluble fraction preparations of healthy and inflamed gingiva from human subjects of both sexes. The subcellular preparations were supplemented with an NADPH-regenerating system and incubated for 2 h at pH 7.4 and 37 degrees C. The metabolites were identified by column, multiple TLC and radioautography and quantified with liquid scintillation counting. In inflamed tissue the metabolic activity was higher than in healthy gingiva. On the basis of the identified metabolites it can be concluded that the human gingiva of both sexes contains marked 3 alpha-, 3 beta- and 20 alpha-hydroxysteroid dehydrogenase, delta 4-5 alpha- and delta 4-5 beta-steroid hydrogenase activities, and less 20 beta-hydroxysteroid dehydrogenase activity.

Adolescent↗

PubMed1986

Role of granulation tissue and fibroblasts in gingival testosterone metabolism in the rat in vitro.

The metabolism of testosterone by experimental granulation tissue, fibroblasts and the oral mucosa of rats of both sexes was studied. The experimental granulation tissue was produced by implanting viscose-cellulose sponges beneath the dorsal skin of female and male rats for 21 days. The granuloma capsules, fibroblasts in the sponges and the oral mucosae were homogenated. Mitochondrial, microsomal and soluble fractions were incubated with [4-14C]testosterone and NADPH for 30 min at pH 7.4 and 37 degrees C. The metabolites were identified with column and TLC and radioautography and quantified with liquid scintillation counting. The experimental granulation tissue and fibroblasts of both sexes showed less activity in metabolizing testosterone than the gingival tissue. The tissues were shown to contain 3 alpha-, 3 beta- and 17 beta-hydroxysteroid dehydrogenase and 5 alpha- and 5 beta-steroid hydrogenase activities. The activities of the enzymes in the oral mucosae were higher than in the experimental granulation tissue and fibroblasts.

Animals↗

PubMed1984

Centripetal spread of endothelial cell mitotic activity in the artery leading to a rapidly growing tumor.

Vascular proliferation and growth have been attributed either to biophysical forces in the vessel lumen or to chemical mediators. In this study growth and endothelial cell tritiated thymidine uptake in the spermatic artery of the rat during early growth of the Walker 256 carcinoma in the testicle were examined. Testicular blood flow was measured with radioactive microspheres; radioautography following administration of tritiated thymidine was employed to define the endothelial cell labeling index; angiography was employed to assess the diameter of the spermatic artery lumen; and the mass of the artery was determined directly. Endothelial proliferation in the spermatic artery was increased on the second day, simultaneous with rather than subsequent to a significant increase in blood flow to the testicle. Moreover, endothelial cell thymidine uptake began to decline well before the blood flow through the artery reached a maximum. A gradient in endothelial cell proliferation and in lumen diameter of the spermatic artery became apparent during tumor growth; the increase in both lumen diameter and endothelial cell thymidine uptake in the spermatic artery occurred first in the portions nearest the testicle and spread centripetally throughout the length of the artery over several days. Because the spermatic artery is an end artery, without branches and without a normal change in internal diameter, and because the increase in blood flow must have occurred throughout the length of the artery at the same time, we conclude that factors other than those related to an increase in blood flow were primarily responsible for the increase in endothelial cell turnover. These observations make a chemical messenger, perhaps moving retrogradely through cell-to-cell contact, the most likely candidate as the responsible mediator.

Animals↗

PubMed1989

Lack of significant changes in mu, delta opioid binding sites and neutral endopeptidase EC 3.4.24.11 in the brain and spinal cord of arthritic rats.

The possible changes in neutral endopeptidase EC 3.4.24.11 ("enkephalinase", NEP), mu and delta opioid binding sites, were investigated using in vitro quantitative radioautography in various regions of the central nervous system of the Freund's adjuvant-induced arthritic rat, a model of chronic pain. Enkephalinase was labeled by a specific tritiated inhibitor, [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly), while mu and delta opioid binding sites were selectively labelled with [3H]Tyr-D-Ala-Gly-(Me)Phe-Gly-ol ([3H]DAGO) and [3H]Tyr-D-Thr-Gly-Phe-Leu-Thr ([3H]DTLFT), respectively. As compared to controls, no significant modifications were found in NEP, mu or delta binding sites at both supraspinal and spinal levels of arthritic rats. These results suggest that the enhanced efficiency of exogenous opioids or endogenous enkephalins, reported to occur in this model of chronic inflammatory pain, are not directly related to changes in mu and delta opioid binding sites or steady state levels of NEP.

Animals↗

PubMed1981

Uptake of tritium-labelled biogenic amines by the prostomium of the polychaete Nereis virens (Sars) (Annelida).

The uptake of tritium-labelled 5-HT, noradrenaline, 5-hydroxytrytophan, DOPA and dopamine by the cerebral ganglion and prostomial nervous system of the polychaete Nereis virens has been examined using radioautography at the level of the light microscope. Pronounced uptake of (3)H-5HT occurred in the antennal, palpal, tegumentary and nuchal nerves as well as in ganglionic nuclei 13, 14, 15, 16, 17, 20, 24 and 25, the mid-brain neuropile, the neurosecretory neuropil and the infracerebral organ; (3)H-NA uptake was observed in small cells in the prostomial epidermis, and the infracerebral organ; (3)H-dopamine only in one of two common types of epidermal mucus cells. Prostomial muscles labelled generally with (3)H-NA and at specific sites with (3)H-5HT. These observations support the concept of an efferent serotonergic system originating in several cerebral ganglionic nuclei and serving prostomial muscle and epidermis. Evidence for an afferent adrenergic system is less substantial. The role of dopamine remains obscure.

5-Hydroxytryptophan↗

PubMed1990

Sequential changes in the nucleoli of human spermatogonia with special reference to rDNA location and transcription.

The nucleoli of human spermatogonia were studied using electron microscopy, silver staining, radioautography and in situ hybridization. In all types of A spermatogonia, nucleoli were consistently located at the periphery of the nucleus and contained a single fibrillar center associated with the nuclear envelope. In B spermatogonia, nucleoli were centrally located in the nuclei and showed several fibrillar centers or were found to disintegrate. Nucleolar morphology was found to be a good, though not an unequivocal indicator of spermatogonial type. The observed changes in nucleolar morphology reflect the differentiation of spermatogonia: the nucleolar disintegration seen in B spermatogonia corresponds to a pre-leptotene cessation of rDNA transcription. In radioautographs following 3H-uridine uptake, the label was consistently found over the dense fibrillar component, except in the B spermatogonia with disintegrating nucleoli, where no uptake could be detected. In situ hybridization demonstrated that the distribution of rDNA did not correspond to the site of the fibrillar center but to the dense fibrillar component. Compared with radioautographs, this finding clearly established that transcribed units of rDNA were located in the dense fibrillar component. Silver staining was strongly positive in fibrillar centers and in the dense fibrillar component. In Ap spermatogonia the silver deposit was often localized at the edge of the fibrillar threads. The relationships between silver-stained proteins and transcribed and nontranscribed portions of ribosomal genes are reevaluated.

Autoradiography↗

PubMed1994

Decline in natural killer cell-mediated immunosurveillance in aging mice--a consequence of reduced cell production and tumor binding capacity.

The mechanism(s) responsible for the functional deficiencies in aging mice of natural killer (NK) cells, the lymphocytes active in early neoplasia development, are unknown. The aim of the present study was to examine the effect of indomethacin, a prostaglandin synthetase inhibitor, and recombinant interleukin-2 (rIL-2), an NK cell stimulant, on the NK cell numbers and function of aging mice. This combination is highly effective in inducing proliferation and activation of the NK cells in young adult mice. Ten- to 13-month-old DBA/2 mice received either indomethacin daily for 9 days in their drinking water, or rIL-2 twice a day for 4 days, or both agents combined. Untreated aging mice had 50% fewer splenic NK cells than did young adult (5-8 weeks) DBA/2 mice, although bone marrow contained NK cell numbers similar to that of young adult mice. Neither indomethacin alone, rIL-2 alone nor the combination of both could induce an increase in NK cell numbers or function in aging mice. Radioautography combined with immunoperoxidase labelling techniques revealed that the production of new NK cells from the bone marrow of aging mice was significantly reduced relative to that of young adult mice. This was reflected in low numbers of newly formed NK cells accumulating in the spleens of aging mice. The target binding capacity, a necessary precytolytic event, of aging mouse NK cells was also reduced compared to that of young adult mice. The results suggest that the functional deficiencies of aging mouse NK cells are a consequence of multiple factors including an absolute loss of NK cells resulting from a reduced production of such cells in the bone marrow and a decreased capacity of NK cells to bind their tumor targets.

Aging↗