The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N- acetylmannosamine. II. Observations in tissues other than liver

Biochemical evidence from the preceding paper indicated that [3H]N- acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N- acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.     

Effect of light on the labeling of optic tectum gangliosides after an intraocular injection of N-[3H]acetylmannosamine.

Axonally transported gangliosides from retina were more labeled in the optic tectum of chickens exposed to light compared to those maintained in the dark. No differences were observed between the labeling of retinal gangliosides from the two groups. These results indicate that light modifies either the labeling of ganglion cell gangliosides or their axonal transport.     

Migration of glycoprotein from the Golgi apparatus to the surface of various cell types as shown by radioautography after labelled fucose injection into rats

A single intravenous injection of L-[³H]fucose, a specific glycoprotein precursor, was given to young 35–45 g rats which were sacrificed at times varying between 2 min and 30 h later. Radioautography of over 50 cell types, including renewing and nonrenewing cells, was carried out for light and electron microscope study. At early time intervals (2–10 min after injection), light microscope radioautography showed a reaction over nearly all cells investigated in the form of a discrete clump of silver grains over the Golgi region. This reaction varied in intensity and duration from cell type to cell type. Electron microscope radioautographs of duodenal villus columnar cells and kidney proximal and distal tubule cells at early time intervals revealed that the silver grains were restricted to Golgi saccules. These observations are interpreted to mean that glycoproteins undergoing synthesis incorporate fucose in the saccules of the Golgi apparatus. Since fucose occurs as a terminal residue in the carbohydrate side chains of glycoproteins, the Golgi saccules would be the site of completion of synthesis of these side chains. At later time intervals, light and electron microscope radioautography demonstrated a decrease in the reaction intensity of the Golgi region, while reactions appeared over other parts of the cells: lysosomes, secretory material, and plasma membrane. The intensity of the reactions observed over the plasma membrane varied considerably in various cell types; furthermore the reactions were restricted to the apical surface in some types, but extended to the whole surface in others. Since the plasma membrane is covered by a "cell coat" composed of the carbohydrate-rich portions of membrane glycoproteins, it is concluded that newly formed glycoproteins, after acquiring fucose in the Golgi apparatus, migrate to the cell surface to contribute to the cell coat. This contribution implies turnover of cell coat glycoproteins, at least in nonrenewing cell types, such as those of kidney tubules. In the young cells of renewing populations, e.g. those of gastro-intestinal epithelia, the new glycoproteins seem to contribute to the growth as well as the turnover of the cell coat. The differences in reactivity among different cell types and cell surfaces imply considerable differences in the turnover rates of the cell coats.     

Influence of colchicine and vinblastine on the intracellular migration of secretory and membrane glycoproteins: I. Inhibition of glycoprotein migration in various rat cell types as shown by light microscope radioautography after injection of 3H-fucose

Previous studies have shown that colchicine and vinblastine inhibit secretion in many cell types by interrupting the normal intracellular migration of secretory products. In the present work, radioautography has been used to study the effects of these drugs on migration of membrane and secretory glycoproteins in a variety of cell types. Young (40 gm) rats were given a single intravenous injection of colchicine (4.0 mg) or vinblastine (2.0 mg). At 10 min after colchicine and 30 min after vinblastine administration, the rats were injected with 3H-fucose. Control rats received 3H-fucose only. All rats were sacrificed 90 min after 3H-fucose injection and their tissues processed for light microscope radioautography. Examination of secretory cell types such as ameloblasts and thyroid follicular cells in control animals revealed reactions of approximately equal intensity over the Golgi region and over extracellular secretion products, while in drug-treated rats most of the reaction was confined to the Golgi region. In a variety of other cell types, including endocrine cells (e.g., hepatocytes) and cells generally considered as nonsecretory (e.g., intestinal columnar cells), reaction in control animals occurred both over the Golgi region and over various portions of the cell surface. In drug-treated animals, a strong Golgi reaction was present, but reaction over the cell surface was weak or absent. These results indicate that in many cell types, colchicine and vinblastine inhibit migration out of the Golgi region not only of secretory glycoproteins, but also of membrane glycoproteins destined for the plasma membrane.     

Trypanosoma cruzi: incorporation of [3H]-palmitic acid and [3H]-galactose into components shed by trypomastigotes.

Trypomastigotes were metabolically labeled with [3H]-palmitic acid or [3H]-galactose and labeled components were detected in the culture medium. Thin layer chromatography of the shed material showed several lipids in the [3H]-palmitic acid labeled sample while the sugar was mainly incorporated into macromolecules. The material incorporated with the lipidic precursor was fractionated by DEAE-Sephadex (acetate form) and the amount of radioactivity was ten times higher in the acidic lipids than in the neutral lipids. When acidic lipids were further separated by Unisil, 73% of the radioactivity was recovered in the less polar fraction. Different patterns were obtained on comparison of the shed components with the lipids remaining in the parasite.     

Effects of carcinogenic halogenated aliphatic hydrocarbons on [3H]thymidine incorporation into various organs of the mouse. A comparison between 1,2-dibromoethane and 1,2-dichloroethane

The effects of 1,2-dibromoethane (DBE) and 1,2-dichloroethane (DCE) on the incorporation of [3H]thymidine into DNA were evaluated in various tissues of mice. The compounds were given intraperitoneally 24 h before sacrifice in an equimolar dose (293 mumoles/kg body weight). 2 h before the animals were killed, 0.5 mu Ci [3H]thymidine/g body weight was injected intraperitoneally. Both agents inhibited the [3H]thymidine incorporation in the forestomach, a site for their carcinogenic action. Whereas DBE also suppressed the [3H]thymidine incorporation in the nasal mucosa, the thymus, and the "glandular stomach", DCE was inhibitory only in the kidney. The observed difference in the effect of DBE and DCE on the thymus had its counterpart in a DBE-induced decrease of acid-insoluble radioactivity, demonstrated with whole-body autoradiography. The results indicate that in vivo screening of [3H]thymidine incorporation into various organs of an intact experimental animal is a sensitive technique for comparing cyto- and/or genotoxic effects of chemicals with a similar chemical structure.     

Utilization by the isolated perfused rat liver of N-acetyl-D-[1-14C]galactosamine and N-[3H]acetyl-D-galactosamine for the biosynthesis of glycoproteins.

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.     

The isolation of a low molecular weight inhibitor of [3H]TdR incorporation into hepatic DNA.

An inhibitor of [³H]TdR incorporation into rat liver DNA has been partially purified from bovine and rat livers and from bovine serum. The material isolated does not contain TdR: it is species non-specific, tissue-specific, and non-cytotoxic, and may contain a hepatic chalone.     

Measles-virus-persistent infection in BGM cells. Modification of the incorporation of [3H]arachidonic acid and [14C]stearic acid into lipids.

In BGM cells chronically infected with measles virus, although the composition of the phospholipids is unaltered, the fatty acid composition is modified. Uninfected, lytic and persistently infected cells were labelled with [3H]arachidonic acid and [14C]stearic acid and their metabolic fate analysed. No difference in the total incorporation was observed in the different systems. However, the [14C]stearic acid and [3H]arachidonic acid were incorporated up to 2-fold and 13-fold respectively greater into the neutral lipid of persistently infected compared with that of uninfected cells. Both radioactive fatty acids were specifically accumulated in the triacylglycerol and non-esterified fatty acids fractions. Lytically infected cells were similar to uninfected cells. Although there was no significant difference in the incorporation of radioactivity into the total phospholipid in either system, there was a large decrease in [3H]arachidonic acid incorporated into phosphatidylethanolamine and to a lesser extent phosphatidylcholine and phosphatidylinositol in persistently infected cells. [14C]Stearic acid incorporation was also reduced in phosphatidylcholine and phosphatidylethanolamine fractions of persistently infected cells.     

Cytochemical and ultrastructural studies concerning the cell coat glycoproteins in normal and transformed human blood lymphocytes: I. Variations of sialic acid containing glycoproteins subsequent to transformation of T and B lymphocytes by various kinds of stimulating agents

This is an ultrastructural study on variations occurring subsequent to blast transformation in the amount and/or topographical distribution of sialic acid end groups carried by the cell coat glycoproteins of human blood T and B lymphocytes as revealed by PTA staining according to the Rambourg technique.The transformation was immediately followed by a transient decrease of the surface labelling in both T- and B-derived lymph-cell lines, irrespective of the nature of the stimulating agents used for induction of transformation.Nevertheless, upon reaching their final cyto-differentiation step, cells of both lines seemed to recover their initial PTA staining patterns observed in the controls.This transient decrease in the relative number of sialic acid end groups during a period of dramatically rapid cell growth and multiplication may most plausibly be interpreted as reflecting an ‘incomplete maturation’ of the newly synthesized cell coat glycoproteins.     

Structure of the agonist-binding site of the nicotinic acetylcholine receptor. [3H]acetylcholine mustard identifies residues in the cation-binding subsite.

To characterize the structure of the agonist-binding site of the Torpedo nicotinic acetylcholine receptor (AChR), we have used [3H]acetylcholine mustard [( 3H]AChM), a reactive analog of acetylcholine, to identify residues contributing to the cation-binding subsite. Reaction of [3H]AChM, in its aziridinium form, with AChR-rich membrane suspensions, resulted initially in reversible, high affinity binding (K approximately 0.3 microM) followed by slow alkylation of the acetylcholine-binding site. Incorporation of label into AChR alpha-subunit was inhibited by agonists and competitive antagonists, but not by noncompetitive antagonists, and reaction with 3 microM [3H]AChM for 2 h resulted in specific alkylation of 0.6% of alpha-subunits. Within the alpha-subunit, greater than 90% of specific incorporation was contained within an 18-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Val-46 and containing N-linked carbohydrate. To identify sites of specific alkylation, [3H]AChM-labeled alpha-subunit was digested with trypsin, and the digests were fractionated by reverse phase high pressure liquid chromatography. Specifically labeled material was recovered within a single peak containing a peptide extending from Leu-80 to Lys-107. NH2-terminal amino acid sequencing revealed specific release of 3H in cycle 14 corresponding to alpha-subunit Tyr-93. Identification of Tyr-93 as the site of alkylation was confirmed by radiosequence analysis utilizing o-phthalaldehyde to establish that the released 3H originated from a peptide containing prolines at residues 2 and 9. Because [3H]AChM contains as its reactive group a positively charged quaternary aziridinium, alpha-subunit Tyr-93 is identified as contributing to the cation-binding domain of the AChR agonist-binding site. The selective reaction of [3H]AChM with tyrosyl rather than acidic side chains indicates the importance of aromatic interactions for the binding of the quaternary ammonium group, and the lack of reaction with the tyrosyl or acidic side chains within alpha 190-200 emphasizes the selective orientation of acetylcholine within its binding site.     

The metabolism of [carboxyl-14C]anthranilic acid. I. The incorporation of radioactivity into NAD+ and NADP+.

A new pathway of NAD+ synthesis from anthranilic acid was found in the livers of rats. Starting from [carboxyl-14C]anthranilic acid, radioactive NAD+ and NADP+ were produced as judged by Dowex-1 X 8-formate column chromatography followed by radiochromatography. Several intermediate compounds, such as quinolinic acid, nicotinic acid mononucleotide, and nicotinic acid adenine dinucleotide were also identified with the aid of various chromatographic techniques. In the experiments with liver microsomal hydroxylation systems, anthranilic acid was converted into not only 5-hydroxyanthranilic acid but also 3-hydroxyanthranilic acid.     

The effect of various concentrations of nucleobases, nucleosides or glutamine on the incorporation of [3H]thymidine into DNA in rat mesenteric-lymph-node lymphocytes stimulated by phytohaemagglutinin.

1. The rate of [3H]thymidine incorporation into DNA was measured in phytohaemagglutinin-stimulated lymph-node lymphocytes of the rat. 2. Addition of nucleobases or nucleosides to culture medium that already contained 0.2 mM-glutamine had a small stimulatory effect on incorporation. At lower concentrations of glutamine, adenosine (even at 1 microM) caused a marked increase in the rate of incorporation. 3. In the absence of added glutamine, addition of nucleosides or nucleobases markedly increased the rate of incorporation: nucleosides were more effective than nucleobases; and the rate of proliferation in the presence of 10 microM-adenosine plus 10 microM-uridine was similar to that in the presence of optimal concentrations of glutamine. 4. The rate of incorporation was dramatically decreased by an inhibitor of the pathway of pyrimidine nucleotide synthesis de novo. Addition of the pyrimidine nucleosides completely overcame the inhibition; addition of the pyrimidine nucleobases was much less effective. 5. These results indicate that, for proliferation of lymphocytes, glutamine is not essential and can be partially or totally replaced by nucleosides and, to some extent, by nucleobases.     

Rate of incorporation of [3H]thymidine in various tissues of the mouse. A basis for the evaluation of genotoxic effects of chemicals

[3H]Thymidine has been extensively used as a selective precursor to DNA in studies on the kinetics of cell proliferation. We have become interested in measuring early inhibition of the DNA synthesis in various organs of intact animals for detecting genotoxic properties of chemicals. Such experiments should, for convenience and to achieve a large capacity, be performed in the simplest way possible. The present paper deals with some practical aspects on the use of [3H]thymidine in vivo. [6-3H]Thymidine was injected intraperitoneally in mice and the uptake of radioactivity was evaluated by using whole-body autoradiography and liquid scintillation spectrometry. Autoradiograms of sections washed with trichloroacetic acid and methanol were compared with those subjected only to freeze-drying. Liquid scintillation counting was performed of total, non-volatile, acid-insoluble and DNA-associated radioactivities. A rapid increase of the [3H]thymidine incorporation was seen during the first hour after the injection. Further prolongation of the survival time did not result in any significant increase of the incorporated radioactivity. Moreover, there were only slight differences between the autoradiograms from extracted and non-extracted sections. Radioactivities associated with DNA closely correlated to those representing acid-insoluble material, indicating that acid-insoluble radioactivity provides a good estimate of the [3H]thymidine incorporation into DNA.     

Biosynthesis of rat sperm outer dense fibers during spermiogenesis. In vivo incorporation of [3H]leucine into the fibrillar complex

The temporal incorporation profile of [3H]leucine into the outer dense fiber polypeptides was determined after the intratesticular injection of the radioisotope. Groups of four rats were killed on alternate days after injection, and the outer dense fibers were isolated from the caput epididymal sperm. The radioactivity incorporated into the whole sperm and into the isolated fibers showed a sharp peak at 10 days after injection. Therefore, considering the known kinetics of spermatogenesis in the rat, the maximal incorporation of radioactivity into the fibers occurred during the second half of spermiogenesis. The radioactivity incorporated into the six major polypeptides of the fibers separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate accounted for 95 percent of the total radioactivity associated with the isolated fibrillar complex. Furthermore, analysis of the time-course incorporation of [3H]leucine into the polypeptides of the fibers indicated that the maximal incorporation into each of the six major components took place within the same period of time. Using two different procedures, the specific activity of each major polypeptide was determined at the time of maximal incorporation. It was found that the specific activity of the most abundant components (molecular weights of 30,400 plus 26,000) was approximately twice that of the other polypeptides.     

The study of the effects of mechanical vibration at infrasound frequency on [3H]-thymidine incorporation into DNA of E. coli K-12

The aim of the present work was to investigate the frequency-dependent effects of mechanical vibration at infrasound frequency (MV at IS frequency or MV) on E. coli K-12 growth by investigating the cell proliferation, using radioactive [³H]-thymidine assay. The frequency-dependent effects of MV were shown that it could either stimulate or inhibit the growth of microbes. However, the mechanism through which the MV effects affect the bacterial cells is not clear yet. It was suggested that the aqua medium can serve as a target through which the biological effect of MV on microbes could be realized. To check this hypothesis the frequency-dependent effect (2, 4, 6, 8, 10 Hz) of MV on the bacterial growth in cases of exposure the preliminary treated microbes-free medium and microbes containing medium were studied. It has been shown that MV at 4, 8, and 10 Hz frequency has inhibition effects, while at 2 and 6 Hz has stimulation effects on cell proliferation.