Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Effect of colchicine on the redistribution of L-[1,5,6-3H]fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes.

To define the role of cytoplasmic microtubules in the biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells, we studied the effect of colchicine on the incorporation of L-[1,5,6-3H]fucose into Golgi, lateral basal and microvillus membranes. Colchicine was administered intraperitoneally before or after injection of radioactive fucose. The incorporation of radioactivity into Golgi membranes was little affected by colchicine, which did not prevent the redistribution of most of the labelled glycoproteins from the Golgi complex into other parts of the villus cell. The incorporation of labelled glycoproteins into the microvillus membrane was greatly inhibited by colchicine given 2 h or 10 min before the radioactive fucose: all labelled glycoproteins present in this membrane were equally affected. In contrast, the administration of colchicine considerably increased the incorporation of radioactivity into the lateral basal part of the plasmalemma, and prevented the disappearance of most of the labelled glycoproteins from this membrane at late times after fucose injection. These results suggest that cytoplasmic microtubular structures are important for the polarization of the intestinal villus cell and the biogenesis of the microvillus membrane, although playing little or no role in the movement of membrane components from the Golgi complex to the lateral basal part of the plasmalemma.     

Increased Fucosylation of Chick Brain Proteins Following Training: Effects of Cycloheximide

When chicks are trained to avoid pecking a bead coated with methylanthranilate in a one-trial passive avoidance task there is an increase in fucose incorporation in vivo and in vitro in the right forebrain base of methylanthranilate (M)-trained compared to water (W)-trained chicks. The relation of this increase to de novo protein synthesis in vivo and in vitro has been examined. Cycloheximide (Cx), 1 mM, inhibited in vitro fucosylation of chick brain slices by 60% after 3 h. However, the training-related increase in in vitro fucosylation still persisted. When Cx was injected intraventricularly 10 min before training, the subsequent increase in in vitro fucosylation due to training was still apparent. When Cx was injected and [14C]leucine and [3H]fucose incorporation studied in vivo in M-trained and W-trained chicks, there was no increase in fucosylation due to training in the Cx-treated M-trained over the W-trained chicks. These results are taken to indicate that in vitro fucosylation and its increase subsequent to training is not protein synthesis-dependent, but that both in vivo and in vitro there are interactions between Cx and fucosylation steps that are independent of Cx's effects on protein synthesis.     

Rapidly metabolized glycoproteins in a neuroplastoma cell line

The metabolism of neuroblastoma cell glycoproteins was examined using l-[³H]fucose. Incubation of monolayer cultures with [³H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [³H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h)_ of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [³H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 · 10^?4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [³H]fucose. Although the major proportion of the radioactivity remained as [³H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components. However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [⁴C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.     

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose

Summary The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated ³H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.     

Presence of glycoproteins in the cell nucleus as shown by radioautographic studies after administration of [3H]fucose and [3H]galactose

Dorsal root ganglia were removed from adult bullfrogs and incubated with [3H]fucose for intervals from 15 min to 1 h, followed by fixation. Some ganglia were post-incubated in the absence of [3H]fucose for up to 17 h. In additional in vivo experiments, young frogs were injected with [3H]fucose, and killed 30 min or 1 h later, and then ganglia were removed and fixed. Electron microscope radioautographs of the ganglia revealed an intense radioautographic reaction over the nuclei of Schwann and satellite cells as early as 5 min after initial exposure to [3H]fucose. At time intervals up to 2 h after initial exposure to [3H]fucose, the silver grains were evenly distributed over both the periphery and internal regions of the nucleus, while at 18 h they were localized to the cell periphery. In occasional cells, the perinuclear space was expanded in some areas and was the site of reaction. In young rats, injected with [3H]galactose and killed 15 min to 5 h later, electron microscope radioautographs revealed heavy reaction over the nuclei of duodenal villous and crypt columnar cells, in which the grains were evenly distributed over both the peripheral and internal regions. In mitotic cells, grains appeared to be associated with the condensed chromatin of forming chromosomes. These results provide strong evidence that glycoproteins exist in the nuclei of the above cell types and that they are actively renewed. The rapid appearance of nuclear reaction after initial exposure to [3H]fucose or [3H]galactose indicates that either these sugars are added to glycoproteins within the nucleus itself or that they migrate rapidly to this site after having been glycosylated elsewhere.     

AUTORADIOGRAPHIC STUDIES OF SYNTHESIS AND INTRACELLULAR MIGRATION OF GLYCOPROTEINS IN THE RAT ANTERIOR PITUITARY GLAND

The incorporation of [³H]fucose in the somatotrophic and gonadotrophic cells of the rat adenohypophysis has been studied by electron microscope autoradiography to determine the site of synthesis of glycoproteins and to follow the migration of newly synthesized glycoproteins. The pituitaries were fixed 5 min, 20 min, 1 h, and 4 h after the in vivo injection of [³H]fucose and autoradiographs analyzed quantitatively. At 5 min after [³H]fucose administration, 80–90% of the silver grains were localized over the Golgi apparatus in both somatotrophs and gonadotrophs. By 20 min, the Golgi apparatus was still labeled and some radioactivity appeared over granules. At 1 h and 4 h, silver grains were found predominantly over secretory granules. The kinetic analysis showed that in both protein-secreting cells (somatotrophs) and glycoprotein-secreting cells (gonadotrophs), the glycoproteins have their synthesis completed in the Golgi apparatus and migrate subsequently to the secretory granules. It is concluded from these in vivo studies that glycoproteins which are not hormones are utilized for the formation of the matrix and/or of the membrane of the secretory granules. The incorporation of [³H]fucose in gonadectomy cells (hyperstimulated gonadotrophs) was also studied in vitro after pulse labeling of pituitary fragments in medium containing [³H]fucose. The incorporation of [³H]fucose was localized in both the rough endoplasmic reticulum (ER) and the Golgi apparatus. Later, the radioactivity over granules increased while that over the Golgi apparatus decreased. The concentration of silver grains over the dilated cisternae of the rough ER was not found to be modified at the longest time intervals studied.     

Intracisternal neurotoxins and monoamine neurons innervating the spinal cord: Acute and chronic effects on cell and axon counts and nerve terminal densities

Summary The total number of catecholamine (CA) and 5-hydroxy-trydroxy-tryptamine (5-HT) containing nerve cell bodies in groups A1-7 and B1-3 were counted using Falck-Hillarp fluorescence histochemistry and found to be about 5000 and about 6300 respectively. The total number of CA axons in the white matter of the spinal cord was found to be about 4300 at the cervical level and about 3300 at the lumbar level as revealed in cryostat sections. Together with previous studies our quantitative results suggest that individual cell bodies in locus coeruleus send axon collaterals to the spinal cord, cortex cerebri, cortex cerebelli and probably also other areas of the brain.The decrease in number of CA axons in spinal cord occurred approximately at the level where the CA nerve terminal innervation of the sympathetic lateral column ends. Single CA axons were observed to give off multiple branches at right angles, suggesting that single CA axons innervate several different levels of the grey matter.A transient loss of visible CA cell bodies in groups A1-7 was observed after intracisternal injection of 25–50 g 6-hydroxydopamine (6-OH-DA). A permanent loss of visible 5-HT cell bodies in groups B1-3 was observed after 25 g 5,6-dihydroxy-tryptamine (5,6-HT) given intracisternally, a transient decrease was obtained when the same dose of 5,6-HT was given intraventricularly.CA nerve terminals regenerated in a cranio-caudal direction along the motoneuron columns after lesioning with 6-OH-DA given intracisternally. The reappearing CA nerve terminals seemed to outline the longitudinally oriented dendritic bundles of the motoneurons and was exclusively found in these areas of the ventral horn. Thus, no substantial CA nerve terminal reinnervation of zona intermedia or the dorsal horn was observed. About 10% of the CA axons running in white matter were left after intracisternal 6-OH-DA and found at all levels of the cord. The number of remaining CA axons did not increase with time.After 10–50 g 5,6-HT given intracisternally a return of 5-HT nerve terminals was only observed in cervical segments. There was a 60–90% decrease in number of axons that accumulated 5-HT after an acute transverse lesion and no recovery of the number of such accumulations was observed during the following six months.It was concluded that regeneration of CA nerve terminals in the spinal cord can occur after lesioning with 6-OH-DA given intracisternally. Regeneration of 5-HT nerve terminals following intracisternal 5,6-HT is very restricted probably because of the permanent loss of 5-HT cell bodies obtained by the intracisternal route of administration as opposed to the intraventricular route. It was suggested that the reappearing CA nerve terminals were derived from locus coeruleus and that they were specifically guided by the motoneuron columns.     

Pinocytosis in mouse L-fibroblasts: ultrastructural evidence for a direct membrane shuttle between the plasma membrane and the lysosomal compartment

Mouse L-fibroblasts internalized large amounts of cationized ferritin (CF) by pinocytosis. Initially (60-90 s after addition of CF to cell monolayers at 37 degrees C), CF was found in vesicles measuring 100-400 nm (sectioned diameter) and as small clusters adhering to the inner aspect of the limiting membrane of a few large (greater than 600 nm) vacuoles. After 5-30 min, CF labeling of large vacuoles was pronounced and continuous. Moreover, 70-80% of all labeled structures were tiny (less than 100 nm) vesicles. However, the absolute frequency of tiny vesicles increased more than twofold from 5 min to 30 min. When the cells were incubated with CF for 30 min, then washed and further incubated for 3 h without CF, almost all CF was present in dense bodies (100-500 nm). When L-cells were first incubated with horseradish peroxidase (HRP), then washed and incubated with CF, double-labeled vacuoles were observed. Tiny vesicles also contained HRP-CF, and small HRP-CF patches were localized on the cell surface. Distinct labeling of stacked Golgi cisterns was not observed in any experiment. These observations suggest that the numerous tiny vesicles are not endocytic but rather pinch off from the large vacuoles and move towards the cell surface to fuse with the plasma membrane. Thus, ultrastructural evidence is provided in favor of a direct membrane shuttle between the plasma membrane and the lysosomal compartment.     

Retrograde axonal transport after radioactive serotonin injections into the olfactory bulb: a biochemical analysis of transported radioactive material

A biochemical analysis of radioactive compounds was performed in the olfactory bulb (OB) and raphe dorsalis (RD) after injection of radioactive [³H] or [¹⁴C]serotonin (5-HT ranging from 10^?2 M to 10^?7 M) into the OB of rats treated or not with a monoamine-oxidase inhibitor (MAOI).In the OB of untreated rats, radioactivity was associated with precipitated protein and soluble perchloric acid (PCA) fractions. High performance liquid chromatography (HPLC) analysis of the PCA-supernatant gave 4 radioactive peaks: one associated with endogenous 5-HT, another with endogenous 5-hydroxyindole acetic acid (5-HIAA) and two without any relationship with endogenous hydroxyindoles: a ‘5-HT derivative A’ and a ‘5-HT derivative B’. The presence of these ‘5-HT derivatives’ was significantly reduced after treatment with 5,6-dihydroxytryptamine.In the RD, radioactivity was associated with the protein fraction and with ‘5-HT derivative A’. The kinetic analysis (from 30 min to 46 h) of the ‘5-HT derivative A’ was characterized by a disappearance in the OB and an accumulation in the RD corresponding to a rate of migration in a range of 0.7 to 2 mm/h. This compound was absent or negligible in other non-serotoninergic neurons (such as the Locus Coeruleus, Amygdala and Cortex piriformis). No clear evidence for retrograde transport of radioactive 5-hydroxytryptophan (5-HTP) or 5-HIAA was found.At lower concentration of 5-HT injected into the OB, the half lives and the times of maximal accumulation for 5-HIAA, ‘5-HT derivative A’ and ‘5-HT derivative B’ were increased. The specific activity of 5-HT and 5-HIAA was also increased.The selective radioactive accumulation in the cell bodies of RD neurons after injection of radioactive 5-HT into the OB is discussed as resulting from a selectivity in (a) the uptake by 5-HT nerve terminals; (b) the metabolism of 5-HT into ‘5-HT derivative A’ in the OB; (c) the retrograde axonal transport of ‘5-HT derivative A’. This ‘5-HT derivative A’ could represent a messenger between nerve terminals and cell bodies and could be involved in homeostatic mechanisms that maintain cellular dynamics.When a MAOI was used, ‘5-HT-derivative A’ and [³H]5-HT were found in the OB and also in the RD cell bodies, and to a lesser extent, in the non-serotoninergic cell bodies. These results indicate that MAO inhibition produces a relative non-selectivity in the ‘uptake-metabolism and retrograde axonal transport’ systems.     

Retrograde axonal transport specificity in the locus coeruleus neurons after [H]noradrenaline injection into the rat olfactory bulb

Retrograde axonal transport process was investigated in the afferent systems to the rat olfactory bulb, after [³H]noradrenaline ([³H]NA) injection into the olfactory bulb, in order to provide evidence regarding its specificity and the biochemical basis supporting this specificity.

Radioautographs showed that [³H]NA unilaterally injected into the olfactory bulb at a concentration of 10⁻³ M, resulted in labeling of the structures afferent to the olfactory bulb, mainly on the injected side: locus coeruleus (LC), dorsal and central raphes, nucleus of the lateral olfactory tract and piriform cortex. Heavy labeling was observed on the noradrenergic LC cell bodies, whereas the radioautographic reaction was less intense on the other structures. After 10⁻⁴ M injection, the labeling intensity of the LC cell bodies was lower while very rare weakly labeled cell bodies persisted in the dorsal raphe, nucleus of the lateral olfactory tract and piriform cortex. The LC cell bodies were exclusively labeled when the concentration of [³H]NA injection was 10⁻⁵ M. All the other structures were devoid of labeling. It was still possible to detect labeled cell bodies in the LC for a 10⁻⁶ M concentration.

Following bilateral injections of [³H]NA (10⁻³ M) the total radioactivity retrogradely transported to the LC represented about 4 times the total radioactivity measured in the periaqueductal gray substance (as control tissue of the tracer diffusion). Fractional study by ethanol of LC tissue homogenate and liquid scintillation counting of each fraction showed that 60% of the total radioactivity (about 2.5 times the control value) was in the supernatant and 40% (about 20 times the control value) was associated with the precipitate. In the other regions such as the dorsal and central raphes and periaqueductal gray substance, radioactivity was mainly found in the supernatant, except for the dorsal raphe whose precipitate contained a low amount of radioactivity (about 4 times the control value).

Colchicine (an axonal transport inhibitor) bilaterally injected into the medial forebrain bundle and systemic administration of desipramine (a noradrenaline uptake inhibitor) decreased the radioactivity associated with the LC precipitate by 90 and 85% and the LC supernatant radioactivity by 55 and 35%, respectively. These pretreatments did not significantly affect the radioactivity amounts measured in the different fractions of dorsal and central raphes and periaqueductal gray substance. Radioautographic study after colchicine treatment showed a large decrease in the labeling intensity of the LC cell bodies as compared to the non-treated side.

Therefore, we suggest that low concentrations (10⁻⁵ M) of [³H]NA injected in the olfactory bulb determine specific conditions of noradrenergic pathway labeling. This specific labeling after migration could be supported by the radioactive ethanol precipitate which would appear to contain [³H]NA- and/or ³H-derivatives-binding protein. Such a ³H-macromolecular complex, which could represent the specific carrier, may well undergo retrograde transport from the nerve terminals towards the cell bodies.     

The effect of glycoprotein-processing inhibitors on fucosylation of glycoproteins

The influenza viral hemagglutinin contains L-fucose linked alpha 1,6 to some of the innermost GlcNAc residues of the complex oligosaccharides. In order to determine what structural features of the oligosaccharide were required for fucosylation or where in the processing pathway fucosylation occurred, influenza virus-infected MDCK cells were incubated in the presence of various inhibitors of glycoprotein processing to stop trimming at different points. After several hours of incubation with the inhibitors, [5,6-3H]fucose and [1-14C]mannose were added to label the glycoproteins, and cells were incubated in inhibitor and isotope for about 40 h to produce mature virus. Glycopeptides were prepared from the viral and the cellular glycoproteins, and these glycopeptides were isolated by gel filtration on Bio-Gel P-4. The glycopeptides were then digested with endo-beta-N-acetylglucosaminidase H and rechromatographed on the Bio-Gel column. In the presence of castanospermine or 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine, both inhibitors of glucosidase I, most of the radioactive mannose was found in Glc3Man7-9GlcNAc structures, and these did not contain radioactive fucose. In the presence of deoxymannojirimycin, an inhibitor of mannosidase I, most of the [14C]mannose was in a Man9GlcNAc structure which was also not fucosylated. However, in the presence of swainsonine, an inhibitor of mannosidase II, the [14C]mannose was mostly in hybrid types of oligosaccharides, and these structures also contained radioactive fucose. Treatment of the hybrid structures with endoglucosaminidase H released the [3H]fucose as a small peptide (Fuc-GlcNAc-peptide), whereas the [14C]mannose remained with the oligosaccharide. The data support the conclusion that the addition of fucose linked alpha 1,6 to the asparagine-linked GlcNAc is dependent upon the presence of a beta 1,2-GlcNAc residue on the alpha 1,3-mannose branch of the core structure.     

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose

The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated 3H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.     

Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

Epithelial repair is inhibited by an alpha(1,6)-fucose binding lectin

The effective repair of damage to the airway epithelium is essential to maintain the ability to exclude airborne particulates and protect against potential pathogens. Carbohydrates on the cell surface have an important role in cell-cell and cell substrate interactions. Using a model of repair with airway epithelial-derived cells of the 16HBE 14o(-) cell line, we have examined the effect of the Aleuria aurantia lectin (AAL), which binds very selectively to alpha(1,6)-linked fucose residues. Addition of unconjugated or FITC-labeled AAL reduced the rate of epithelial repair to approximately one-third of control values as measured by image analysis while cell viability was maintained. Pulse labeling with AAL-FITC for 30 min followed by incubation in AAL-free medium caused similar inhibition of repair but could be reversed by addition of fucose up to 7 h after AAL removal. By confocal microscopy, AAL binding was found to be on the apical, but not basolateral, surfaces of cells, and internalization of the labeled lectin was seen. Preincubation of the lectin with fucose prevented this effect. Ulex europeaus I lectin, which is also fucose specific, resulted in similar binding to the cells and internalization, but it did not affect the speed of the repair process. We conclude that alpha(1,6)-fucose binding sites play an important role in epithelial repair. Better understanding of this process will provide a deeper insight into the crucial mechanisms of epithelial repair.     

Dexamethasone and corticosterone receptor sites. Differential topographic distribution in rat hippocampus revealed by high resolution autoradiography

High resolution light microscopic autoradiography was used, together with regional surveys and combined acridine orange staining, to define in rat hippocampus cellular and subcellular sites of concentration and retention of 3H dexamethasone and to compare the topographic pattern of labeling with that of 3H corticosterone. Nuclear uptake of 3H dexamethasone in the hippocampus is demonstrated for the first time in vivo. With 3H dexamethasone, strongest nuclear radioactive labeling was observed in certain glial cells throughout the hippocampus, followed by strong nuclear labeling in most neurons in area CA1 and in the adjacent dorsolateral subiculum and weak nuclear labeling in granule cells of the dentate gyrus. Neurons in areas CA2, CA3, CA4, and in the dorsomedial subiculum and indusium griseum showed little or no nuclear labeling after 3H dexamethasone. With 3H corticosterone, strongest nuclear labeling was observed in neurons in area CA2 and in the dorsomedial subiculum and indusium griseum, followed by area CA1, then CA3 and CA4; the dentate gyrus contained scattered strongly labeled cells among cells with intermediate nuclear labeling. At the subcellular level, evidence for both nuclear and cytoplasmic accumulation of label was found. The results indicate that dexamethasone and corticosterone have both nuclear and cytoplasmic binding sites and that particular patterns of target cell distribution exist, characteristic for each agent. This suggests a differential regulation of cellular functions for the two compounds. Corticosterone nuclear binding appears to be more extensive and encompasses regions with dexamethasone binding. Whether in certain of these common regions corticosterone binds to the same receptor as dexamethasone, which seems possible, or to different receptors, remains to be clarified.     

Age-dependent acute interference with stem and progenitor cell proliferation in the hippocampus after exposure to 1800 MHz electromagnetic radiation

To investigate the effects of exposure to an 1800 MHz electromagnetic field on cell death and cell proliferation in the developing brain, postnatal day 7 (P7) and P21 healthy Kunming mice were randomly assigned into the experimental and control groups. The experimental groups were exposed to an 1800 MHz electromagnetic field for 8 h daily for three consecutive days. The thymidine analog 5-bromo-2-deoxyuridine (BrdU) was injected intraperitoneally 1 h before each exposure session, and all animals were sacrificed 24 h after the last exposure. Cell death and proliferation markers were detected by immunohistochemistry in the dentate gyrus of the hippocampus. Electromagnetic exposure has no influence on cell death in the dentate gyrus of the hippocampus in P7 and P21 mice as indicated by active caspase-3 immunostaining and Fluoro-Jade labeling. The basal cell proliferation in the hippocampus was higher in P7 than in P21 mice as indicated by the number of cells labeled with BrdU and by immunohistochemical staining for phosphor-histone H3 (PHH3) and brain lipid-binding protein (BLBP). Electromagnetic exposure stimulated DNA synthesis in P7 neural stem and progenitor cells, but reduced cell division and the total number of stem cells in the hippocampus as indicated by increased BrdU labeling and reduced PHH3 and BLBP labeling compared to P7 control mice. There were no significant changes in cell proliferation in P21 mice after exposure to the electromagnetic field. These results indicate that interference with stem cell proliferation upon short-term exposure to an 1800 MHz electromagnetic field depends on the developmental stage of the brain.     

Differentiation of cultured epidermal keratinocytes related to sterol metabolism and its retardation by chemical carcinogens

The amount of 5 beta-cholest-7-en-3 beta-ol of mouse dorsal skin was increased after parturition until 10 days of age, reaching a maximum 5 beta-cholest-7-en-3 beta-ol/5-cholesten-3 beta-ol ratio of 0.43. [2-14C]Acetate was incorporated almost exclusively into 5-cholesten-3 beta-ol in the basal cell culture of epidermal keratinocytes. However, when the concentration of calcium was changed from 0.07 to 1.9 mM to induce terminal differentiation of keratinocytes, a definite amount of radioactive acetate was incorporated into 5 beta-cholest-7-en-3 beta-ol. The extent of the incorporation was increased at least until 72 h after changing medium, and the ratio of radioactive 5 beta-cholest-7-en-3 beta-ol/radioactive 5-cholesten-3 beta-ol was constantly increased in this period, indicating that the accumulation of 5 beta-cholest-7-en-3 beta-ol in the cell is concomitant with the differentiation of the cell. Pretreatment with chemical carcinogens such as 7,12-dimethylbenz[a]anthracene and 20-methylcholanthrene inhibited the incorporation of radioactive acetate into 5 beta-cholest-7-en-3 beta-ol in the high calcium medium at least for the initial 24 h. After that, the incorporation was gradually restored to the normal level. Pretreatment with a tumor promoter, such as 12-O-tetradecanoyl-phorbol 13-acetate, however, did not inhibit the incorporation. Thus, sterol metabolism is suggested to be a useful indicator for differentiation of epidermal keratinocytes.     

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

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

The removal of cell surface material by enzymes used to dissociate mammary-gland cells

Summary Treatment of mouse mammary epithelial cells (MMEC) with various enzymes used for dispersing and transferring cells results in extensive digestion of materials on the cell surfaces. MMEC biosynthetially labeled with [³H]fucose, [¹⁴C]fucose and [³H]amino acids or with¹²⁵I by the lactoperoxidase method were exposed to either collagenase plus hyaluronidase, followed by pronase, or to trypsin in concentrations and conditions currently used for cell dispersion. Whereas the latter enzyme preparation solubilized 76% of the trichloroacetic acid precipitable radioactive fucose and 96% of the protein-bound¹²⁵I, collagenase plus hyaluronidase treatment released lesser amounts of each label. Subsequent treatment of the cells with pronase removed additional surface-labeled materials, but the total amounts released were still less than when the trypsin preparation alone was employed. Released cell surface materials were analyzed by gel chromatography. Some of the peaks obtained also were examined by polyacrylamide gel electrophoresis. The labeled materials that remained attached to the MMEC after enzymatic treatment were investigated by these two methods as well. We could show that collagenase plus hyaluronidase solubilized three main glycoprotein components from the cell surface. In addition, we could show that the extensive cell surface damage caused by these two enzyme preparations was due to the high proteolytic activity present in these preparations as judged by their ability to hydrolyze rabbit gamma globulin labeled with¹²⁵I. Even though their membranes were extensively damaged by the enzyme treatments, the dispersed cells could be cultured successfully in vitro and could incorporated fucose into their surfaces in a manner similar to that by intact tissue. Through the use of gel-filtration (cochromatography of [¹⁴C]fucose and [³H]fucose cell surface materials), we could demonstrate the identity of cell surface glycoproteins synthesized by cultured cells and by intact tissue. This work was supported by Grant Nos. CA 11736 and CA 19455 from the National Cancer Institute, and Biomedical Research Support Grant No. RR05467 from the National Institutes of Health, DHEW.