Endocytosis of exogenous GM1 ganglioside and cholera toxin by neuroblastoma cells.

Cholera toxin (CT) covalently linked to horseradish peroxidase (HRP) is a specific cytochemical marker for its receptor, the monosialoganglioside GM1. The binding and endocytosis of exogenous [3H]GM1 by cultured murine neuroblastoma cells (line 2A [CCl-131] ), which contain predominantly GM3, was examined by quantitative electron microscope autoradiography. The relationship between exogenous receptor, [3H]GM1, and CT HRP was studied in double labeling experiments consisting of autoradiographic demonstration of [3H]GM1 and cytochemical visualization of HRP. Exogenous [3H]GM1 was not degraded after its endocytosis by cells for 2 h at 37 degrees C. Quantitative studies showed similar grain density distributions in cells treated with [3H]GM1 alone and in cells treated with [3H]GM1 followed by CT-HRP. Qualitative studies conducted in double labeling experiments showed autoradiographic grains over the peroxidase-stained plasma membrane, lysosomes, and vesicles at the trans aspect of the Golgi apparatus. The findings indicate that exogenous glycolipid is associated with the plasmid membrane of deficient cells and undergoes endocytosis. The quantitative ultra-structural autoradiographic studies are consistent with the hypothesis that the spontaneous endocytosis of exogenous [3H]GM1 controls the subsequent uptake of CT-HRP.     

Glycosylation process in gonadotrophs: a quantitative electron microscope autoradiographic study with labeled glucosamine

Incorporation of [3H]glucosamine into dispersed anterior pituitary cells was studied by electron microscope autoradiography. Gonadotrophs were examined to determine the intracellular route and kinetic patterns of glycosylation. Studies were performed with cells from; (a) normal adult male rats; (b) rats orchidectomized 3 wk earlier; and (c) orchidectomized rats treated with tunicamycin. Our results show that incorporation of [3H]glucosamine first occurs in the rough endoplasmic reticulum (RER), then proceeds in the Golgi elements (where peripheral carbohydrates are attached). Treatment with tunicamycin results in a decrease in labeling of these 2 organelles. Comparison of the kinetic patterns in normal and castrated male rats shows that the accumulation of labeled glycosylated proteins in granules reaches a plateau within 2 hr post-pulse in normal rats, and rises during a 6-hr chase in castrated rats. However, because of the necessity for a rather long 15 min pulse, we cannot exclude the possibility that incorporation of glucosamine during the pulse may occur concomitantly in the RER and the Golgi saccules, to be followed by rapid transfer to the secretory granules.     

Intracellular distribution of GABA in the rat anterior pituitary. An electron microscopic autoradiographic study

We studied the internalization and intracellular distribution of [3H] GABA in rat anterior pituitary cells. Electron microscopic autoradiography of anterior pituitary fragments or dispersed pituitary cells incubated with [3H] GABA showed that lactotrophs and, to a lesser extent, somatotrophs were the only cells that contained radioactive grains. Grain density analysis performed on dispersed pituitary cells after a pulse-chase experiment (10 min pulse and then change to a medium without radioactive GABA for various periods up to 2 h) revealed that GABA internalized by lactotrophs was distributed in various intracellular membranous organelles. Of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria and secretory granules had different time-dependent labeling patterns. The highest grain density values were associated with plasma membrane (at the first chase time) and the Golgi apparatus. Mitochondria and secretory granules also showed significant grain density values. A similar pattern of distribution was observed when fragments of prolactin-secreting pituitary adenomas were incubated with [3H] GABA. These results provide morphological data on the cellular specificity and intracellular distribution of GABA in anterior pituitary cells.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.     

Giardia lamblia: autoradiographic analysis of nuclear replication

Giardia lamblia trophozoites, grown in axenic culture, were labeled for various periods of time with [3H]thymidine. After autoradiography, grains were counted over each of the two nuclei in each trophozoite. Analysis of the fraction of trophozoites labeled for each time period resulted in an estimate of a generation time of 15 hr. The DNA synthetic or S phase for a trophozoite in culture was calculated to be 1.8 hr. G1 and G2 periods were determined to be 8.5 and 3 hr, respectively. A comparison of the labeling density between the two nuclei indicated that replication takes place simultaneously in both nuclei for at least 70% of S period. The fraction of asymmetrically labeled trophozoites is consistent with a model in which the nuclei replicate out of phase by 15-30 min, but, due to the small diameter of the nuclei relative to the grain size, the possibility that replication takes place simultaneously in both nuclei of a trophozoite throughout the S phase cannot be ruled out.     

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.     

Distribution of epidermal growth factor binding sites in the adult rat anterior pituitary gland

The distribution of epidermal growth (EGF) binding sites was studied in the pituitary gland using light and electron microscope autoradiography which was performed at different time intervals (2 to 60 min) after intravenous (IV) injection of [125I]EGF into adult rats. At the light microscopic level, the labeling was found over cells of the anterior pituitary gland. The time-course study performed by light microscope autoradiography showed that the maximal values were reached at the 2 min time interval. At this time interval, most silver grains were found at the periphery of the target cells. After, the number of silver grains decreased progressively and the localization of silver grains in the cytoplasm indicated the internalization of [125I]EGF. Electron microscope autoradiography showed that labeling was mostly restricted to mammotrophs and somatotrophs. Control experiments indicated that the autoradiographic labeling was due specific interaction of [125I]EGF with its binding site. These results indicate that EGF binding sites are present in at least two anterior pituitary cell types and suggest that EGF can exert a physiological role in the pituitary gland.     

QUANTITATIVE AUTORADIOGRAPHIC STUDY OF LABELED RNA IN RABBIT OPTIC NERVE AFTER INTRAOCULAR INJECTION OF [3H]URIDINE

The distribution of labeled RNA in the optic nerve of the rabbit was studied by quantitative ultrastructural autoradiography after the intraocular injection of [³H]uridine. The highest density of silver grains related to [³H]RNA (27–40 grains/100 µm²) was found in glial cell perikarya; a slightly lower density was present in the glial nuclei (19–20 grains/100 µm²). Axons (4–5 grains/100 µm²) and myelin (2–3 grains/100 µm²) had the lowest grain densities. 74–83% of all counted grains were located outside the axons. By comparing the grain density distribution over the axon with that expected in the case of an exclusive labeling of the surrounding myelin and glial cell processes, it was concluded that the axons contained a number of grains representing [³H]RNA significantly higher than that expected to scatter from myelin and glial processes. Most of these grains were concentrated at the periphery of the axon and were not related to axonal mitochondria.     

Immunohistochemical identification of proliferating cells in organ culture using bromodeoxyuridine and a monoclonal antibody

The ability to measure cell proliferation is important in the study of cancer biology. The usual technique for quantitating proliferating cells in tissue explant and organ culture by detection of [3H]-thymidine incorporation into DNA by autoradiography is tedious and time-consuming. We have developed a technique for identification and quantitation of bromodeoxyuridine (an analogue of thymidine) in cultured tissue explants. Fetal mouse colon explants were exposed in vitro to bromodeoxyuridine (BUdR) or [3H]-thymidine for 3 to 72 hr and then for various periods to unlabeled thymidine. The tissues were stained with a monoclonal anti-bromodeoxyuridine antibody and in parallel [3H]-thymidine incorporation was detected by autoradiography. Incorporation of BUdR was measured by quantitating the amount of pigment deposited over nuclei after immunohistochemical staining, using an optical data digitizer. It was found that both techniques identified proliferating cells. Dividing cells were present both in crypts and in the surrounding stroma in Day 14 fetal mouse colon cultures. The immunohistochemical technique was more rapid and less cumbersome than autoradiography.     

Medullary bone osteogenesis following estrogen administration to mature male Japanese quail

The osteogenesis of medullary bone on endosteal bone surfaces of mature male Japanese quail was induced by estradiol valerate (EV) and the sequential changes were characterized by histology, autoradiography, microradiography, and electron microscopy. In untreated controls, endosteal surfaces of the femoral diaphysis were generally inactive and lined by low-density populations of flat bone-lining cells. Within 24 hr of EV administration the surfaces were lined by plump cells with abundant polyribosomes and with large oval to round nuclei. There was an increase in the concentration of [³H]proline near some endosteal surfaces at this time. By 36 hr the developing osteoblasts had extensive rough endoplasmic reticulum (RER) and Golgi complexes. Extracellular matrix with isotropically arranged collagenous fibers was evident. By 48 hr small trabeculae had formed and some of the matrix was beginning to mineralize. Osteoblasts contained abundant dilated RER, many had numerous cell processes and some were becoming surrounded by bone matrix forming osteocytes. From 72 to 120 hr the developing bone grew rapidly from endosteal surfaces into the marrow space. Medullary bone development was accompanied by rapid and dramatic increases in total plasma calcium levels. This study demonstrates a well-defined rapid sequence of induced osteogenesis in vivo and suggests that the bone-lining cell in the postfetal organism may have osteogenic potential.     

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.     

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.     

3H]thymidine uptake in cells of rat condylar cartilage

Weanling rats were injected intraperitoneally with [3H]thymidine and sacrificed from 5 min to 20 days later. Their mandibular condylar cartilages were examined histologically, by thin-layer autoradiography, and by using liquid scintillation and microscopic counting methods. Labeled DNA appeared in some of the chondrocytes of the resting zone as early as 10 min postinjection, and reached the proliferative zone by 24 hr and the hypertrophic zone by 4 days. The labeling pattern in the last zone was more disperse, being oriented toward the periphery of the cells as they became hypertrophic. The maximum number of labeled chondrocytes was reached by 2 hr postinjection. These amounted to approximately 11% of the total chondrocyte population, the majority of which were located in the resting zone (73%). It is concluded that, over this period, the mitotic index for these cells is 50-60 per thousand resulting in approximately 100 labeled chondrocytes. In addition, some of the chondroclasts at the erosion front contained labeled DNA as early as 5 min after [3H]thymidine administration. By 10 min, 65% of these cells exhibited one or more labeled nuclei, and the ratio of labeled cells remained high through 20 days. Chondroclasts were seen to contain a diffuse label within their cytoplasm after 5 days. This label was similar to that seen in hypertrophic chondrocytes that had reached the erosion front by that time. Clearly, chondroclasts exhibit nuclear division and do not form from fusion of hypertrophic chondrocytes, although which specific mononuclear cells may act as chondroclast progenitors is not clear. In addition, these multinucleate resorbing cells are capable of ingesting or phagocytizing nuclear remnants from hypertrophic chondrocytes at the eroding face of cartilage.     

Ultrastructural immunocytochemical localization of cyclic AMP-dependent protein kinase regulatory subunits in rat parotid acinar cells

Polyclonal antibodies to types I and II regulatory (R) subunits of cyclic AMP-dependent protein kinase (cA-PK) were utilized in a post-embedding immunogold-labeling procedure to localize these proteins in rat parotid acinar cells. Both RI and RII were present in the nuclei, cytoplasm, rough endoplasmic reticulum (RER), Golgi apparatus, and secretory granules. In the nuclei, gold particles were mainly associated with the heterochromatin. In the cytoplasm, the label was principally found in areas of RER. Most gold particles were located between adjacent RER cisternae or over their membranes and attached ribosomes; occasional particles were also present over the cisternal spaces. Labeling of the Golgi apparatus was significantly greater than background, although it was slightly lower than that over the RER cisternae. In secretory granules, gold particles were present over the granule content; no preferential localization to the granule membrane was observed. Morphometric analysis revealed equivalent labeling intensities for RI and RII in the cytoplasm-RER compartment. Labeling intensities for RII in the nuclei and secretory granules were about 50% greater than in the cytoplasm-RER, and 3 to 4-fold greater than values for RI in these two compartments. Electrophoresis and autoradiography of the postnuclear parotid-tissue fraction, the contents of purified secretory granules and saliva collected from the main excretory duct, after photoaffinity labeling with [32P]-8-azido-cyclic AMP, revealed the presence of R subunits. Predominantly RII was present in the granule contents and saliva, while both RII and RI were present in the cell extracts. Additionally, R subunits were purified from saliva by affinity chromatography on agarose-hexane-cyclic AMP. These findings confirm the localization of cA-PK in parotid cell nuclei and establish the acinar secretory granules as the source of the cyclic AMP-binding proteins in saliva.     

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.     

Selective Labeling of [3H]2-Deoxy-d-Glucose in the Snake Trigeminal System: Basal and Infrared-Stimulated Conditions

[³H]2-Deoxy-d-glucose (2-DG) and high-resolution autoradiography were employed to investigate labeling patterns of the trigeminal and infrared sensory system in acrotaline snake, the pit viper (Trimeresurus flavoviridis). Following intracardiac injection of 9.25 MBq [³H]2-DG, neurons in the nucleus of the lateral descending trigeminal tract (LTTD), nucleus reticularis caloris (RC), nucleus trigemini mesencephalicus, nucleus trigemini motorius, and trigeminal ganglia were labeled in various degrees after the pit organ had been removed (basal condition). This revealed that a higher rate of glucose utilization occurred in these nuclei than in the common sensory trigeminal nuclei, which lacked labeling entirely. When a pit was stimulated periodically with an infrared stimulus for 45 min, the difference in percentage of labeled cells was ipsilaterally increased by 12.84% in large cells of the LITD and by 7.55% in the RC, as compared with the contralateral, basal-condition side. These slight changes indicate a small increase of glucose consumption during infrared reception. On the other hand, the small cells in the LTTD showed labeling that did not change with stimulation, suggesting that 2-DG uptake in inhibitory interneurons is relatively constant.     

Analyses of the interactions between retinoid-binding proteins and embryonal carcinoma cells

[3H]Retinoic acid (RA) and [3H]retinol bind in an unsaturable manner to isolated nuclei from Nulli-SCC1 and PCC4.aza1R embryonal carcinoma (EC) cells. When nuclei are challenged with the same labeled retinoids on their respective binding proteins (CRABP and CRBP), much less binding is observed and the binding is saturable. RA-CRABP does not compete with [3H]retinol-CRBP for binding to specific Nulli-SCC1 nuclear sites, whereas retinol-CRBP (but not apo-CRBP) actually potentiates the binding of [3H]RA-CRABP to these nuclei. The binding of [3H]RA-CRABP and [3H]retinol-CRBP is not dramatically affected by prior removal of the outer nuclear membrane with Triton X-100. However, treatment with the detergent after the binding reaction is complete removes about half of the bound [3H]RA-CRABP and almost all of the bound [3H]retinol-CRBP. We measured specific retinoid-binding activities in nucleoplasmic extracts of Nulli-SCC1 and PCC4.aza1R cells. The only readily detectable specific binding activity in nucleoplasmic extracts from untreated cells was for [3H]retinol in PCC4.aza1R preparations. Nucleoplasmic extracts from Nulli-SCC1 and PCC4.aza1R cells pretreated with RA had considerable levels of specific [3H]RA-binding activity with little or no increase in [3H]retinol binding. By contrast, similar extracts from Nulli-SCC1 cells treated with retinol bound large amounts of both [3H]retinol and [3H]RA. Under the same conditions, PCC4.aza1R extracts also contained [3H]RA-binding activity with no increase in [3H]retinol binding above the high endogenous levels. Although these results might reflect translocation of binding proteins from cytoplasm to nucleus, other interpretations must be considered since we often observed an increase, rather than the expected reduction, in cytoplasmic retinoid-binding protein levels.     

Biosynthesis of rat liver cytochrome P-450 in mitochondria-associated rough endoplasmic reticulum and in rough microsomes in vivo

The hypothesis of a preferential biosynthesis of a major phenobarbital inducible form of hepatic cytochrome P-450 (P-450b) in mitochondria-associated rough endoplasmic reticulum (RERmito) was tested by measuring incorporation rates of [35S]methionine and delta-amino[3H]levulinate into the hemoprotein in adult rats. RERmito, rough microsomes (RM representing RER not associated with mitochondria) and smooth microsomes (SM) were quantitatively isolated from the same homogenate by rate zonal centrifugation and their content of P-450b determined by rocket immunoelectrophoresis. P-450b was isolated by immunoprecipitation from detergent-solubilized membrane fractions. The time course and rate of incorporation of [35S] methionine into immunoprecipitable P-450b of RERmito and of RM were similar at all time points studied (2-15 min) both under conditions of maximal induction (4 injections of phenobarbital in 4 days) and after a single injection of phenobarbital. The incorporation of [35S]methionine into P-450b of SM was slower at early time points (2-8 min) but similar to RERmito and RM after 15 min. In contrast, at short labeling periods (less than 8 min) more delta-amino[3H]levulinate was incorporated into P-450b of RERmito than into P-450b of RM and SM. No significant accumulation of free apocytochrome P-450b was found in either membrane fraction. These data indicate a close coordination of the biosynthesis and assembly of apocytochrome P-450b and its prosthetic heme but do not support the hypothesis of a major functional role of MITO X RER complexes in the synthesis of microsomal cytochrome P-450b.     

Cytological events involved in protein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]leucine incorporation

Electron microscope autoradiography has been used to study protein synthesis in syncytial and cellular trophoblast of term human placental villi incubated in vitro with tritiated leucine ([3H]leu). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15). The results of this study demonstrated that both cellular and syncytial trophoblast have marked capacities for protein synthesis. Cellular trophoblast synthesized protein in both its rough endoplasmic reticulum (RER) and its ground plasm which contained abundant free ribosomes. The vast majority of 3H-proteins remained within the cell, with some of the proteins synthesized ultimately appearing in the nucleus. A small percentage of grains was ultimately associated with the trophoblast basement membrane. In syncytial trophoblast, the RER was the dominant site for protein synthesis. The autoradiographic data suggested that, as in the cellular trophoblast, the vast majority of 3H-proteins synthesized by the syncytial trophoblast remained within the syncytial trophoblast throughout the incubation period. The major portion of [3H]leu-labeling present in the syncytial trophoblast of villi incubated the longest times (4 h+) remained in association with the RER. Labeled proteins did not become concentrated in syncytial trophoblast Golgi apparatus, vesicles, or granules. In contrast to cellular trophoblast, the nuclei in the syncytium did not contain 3H-proteins at any time-point studied.     

Enzymatic repair of O-alkylated thymidine residues in DNA: involvement of a O4-methylthymine-DNA methyltransferase and a O2-methylthymine DNA glycosylase

The distribution and intracellular translocation of AFB1 in various subcellular fractions was investigated in isolated hepatocytes by pulse-chase experiments. After labeling the hepatocytes with [3H]-AFB1 (14.5 nM) for 15 min, the highest concentration of [3H]-AFB1 was found in the cytosolic fraction where 66% was bound noncovalently and 1.5% covalently. The lowest concentration of [3H]-AFB1 was found in the nuclear fraction; 36% and 4.9% were bound noncovalently and covalently respectively. When the [3H]-AFB1 loaded cells were chased with unlabeled AFB1 (1 microM), the radioactivity of [3H]-AFB1 in the cell lysate and cytosolic fraction decreased in time with an apparent rate of elimination (t1/2) of 93 min and 66 min, respectively. The levels of covalently bound AFB1 increased with time and reached a maximum at 60 min in nuclei (270%), and at 120 min in mitochondria (220%) and cytosol (430%) as compared to the zero time. Only in the microsomal fraction was there no significant increase with time in covalently bound AFB1. These results suggest that the toxin after activation by the microsomal mixed function oxidases was either detoxified or transported to other cellular organelles where covalent binding of macromolecules occurred.