Study of RNA synthesis in the livers of aging mice by means of electron microscopic radioautography.

The application of 3H-uridine radioautography results in labeling of the liver cells in which RNA is synthesized at various ages of the mouse. Quantitative changes of RNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. The silver grains were mainly located in the nucleoli and nuclei and a few in the mitochondria and rough surfaced endoplasmic reticulum of almost all of the cell populations at various ages. The number of silver grains in the hepatocyte gradually increased after birth, reached the maximum at 14 days of postnatal age, then decreased to 24 months with aging. The number of silver grains of the euchromatin was more than those of the heterochromatin of the hepatocyte nuclei at various ages. The number of silver grains of the granular components was more than those of the fibrillar components of the hepatocyte nucleoli at various ages. However, the ratio of silver grains among euchromatin, heterochromatin, granular components and fibrillar components remained approximately constant.     

A radioautographic study on RNA synthesis in aging mouse spleen after 3H-uridine labeling in vitro.

EM radioautographic study on RNA synthesis in aging mouse spleen was conducted after 3H-uridine labeling in vitro. The localization of radiolabelled precursor was used to determine the site of RNA synthesis. The site of the radiolabelled uridine uptake was localized in the haematopoietic cells, particularly in the lymphoblasts. In the labelled cells, most of the silver grains were localized in the nucleus, specifically in the euchromatin. Few cytoplasmic organelles such as the mitochondria and endoplasmic reticulum were labelled with 3H-uridine. Silver grains were also observed over the nucleoli. The labeling index was expressed as the percentage of labelled cells over the total number of cells counted. The labeling index increased from day one after birth and progressively until the 14th day. Thereafter, the labeling index decreased gradually until the 10th month. A significant difference of p less than 0.05 was noted. In all the EMRAG analyzed, it was observed that the number of silver grains per cell increased proportionally with the labeling index. The result of the quantitation of the changes in RNA synthesis correlated well with the maturational development/aging of the animal.     

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with ³H-thymidine, ³H-uridine or ³H-leucine, precursors for DNA, RNA and proteins, as well as ³H-glucose, ³H-glucosamine, ³⁵S-sulfuric acid, or ³H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.     

A quantitative electron microscope autoradiographic study on 125I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 degrees C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 degrees or 38 degrees C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 degrees than at 38 degrees C. Co-incubation of 125I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with 125I-hCG. The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 degrees C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 degrees C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of 125I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

A quantitative electron microscope autoradiographic study on I-human choriogonadotropin internalization in bovine luteal slices

Very few silver grains were seen on the cell surface and none intracellularly after incubation for 2 h at 4 °C. However, numerous grains were seen in various subcellular organelles when the tissues were incubated for 2 h at 22 ° or 38 °C. The grain distribution was qualitatively similar, but quantitatively, there were fewer grains at 22 ° than at 38 °C. Co-incubation of ¹²⁵I-hCG with excess unlabelled hCG resulted in the virtual disappearance of silver grains from all the subcellular organelles. Excess unlabelled human luteinizing hormone (but not follicle-stimulating hormone or prolactin) inhibited the appearance of silver grains in luteal tissue. There were no silver grains in bovine liver slices incubated with ¹²⁵I-hCG.The plasma membrane-associated grains progressively decreased, while intracellular organelle-associated grains increased with time at 38 °C. There were no grains in nuclei at 5 min, but they appeared at 10 min and increased until 120 min. After correction for radiation spread by three-step mask analysis, smooth endoplasmic reticulum and mitochondria did not contain any grains. The grain density was the highest in Golgi, followed by lysosomes, rough endoplasmic reticulum, nuclei, and plasma membranes after incubation for 2 h at 38 °C. Thus, the electron microscope autoradiography approach confirmed our biochemical data in the preceding paper (Chegini et al., Exp cell res 151 (1984) 466 [5]) on time, temperature dependency and specificity of ¹²⁵I-hCG internalization, association of internalized hormone with a variety of intracellular organelles, and the highest uptake in Golgi.     

Light and electron microscopic radioautographic studies on macromolecular synthesis in amitotic hepatocytes of aging mice.

The problem on cell divisions whether cells proliferate by mitosis or amitosis has been the heated controversy among many biologists since the late 19th century. We confirmed by extensive experiments since the mid 20th century that all the cells proliferated by mitosis not by amitosis but that amitosis actually existed in some glandular cells such as hepatocytes or pancreatic acinar cells which showed only amitotic nuclear divisions without cytoplasmic division producing binucleate cells in several kinds of experimental animals. We further verified that such amitotic cells did not synthesize macromolecular compounds incorporating macromolecular precursors such as 3H-thymidine for DNA, 3H-uridine for RNA or 3H-leucine for proteins. Recent experiments at the end of 20th century using many groups of aging mice, from fetal day 19 to postnatal month 24, injected with such precursors, amitotic cells and resulting binucleate cells in the hepatocytes were detected by electron microscopic radioautography and compared to mononucleate cells. The results demonstrated that only a few hepatocytes showing amitotic nuclear division were found labelled with the 3 precursors demonstrating DNA, RNA and protein synthesis. However, the numbers of silver grains showing incorporations of labelled precursors in respective amitotic cells were very few. It was clarified that the amitotic cells did not synthesize such macromolecules as mononucleate hepatocytes did. On the other hand, more binucleate cells were found than the amitotic cells. DNA synthesis of mononucleate and binucleate hepatocyte nuclei was observed at perinatal stage and disappeared at adult stage. The labeling index of mononucleate hepatocytes was greater than that of binucleate hepatocytes. RNA and protein syntheses in karyoplasm and cytoplasm in both mononucleate and binucleate cells increased from perinatal stage, reaching the maxima at adult stage then decreased to senescent stage. Grain counts revealed that synthesized RNA and proteins were more in binucleate cells than mononucleate cells at respective aging stages.     

Electron microscopic radioautographic studies on DNA synthesis in the hepatocytes of aging mice as observed by image analysis

Quantitative changes of DNA synthesis in the hepatocytes of aging mice were studied by electron microscopic radioautography. Ultrastructural changes of labelled and unlabelled hepatocytes were estimated quantitatively by the image analysis. The results revealed that at various ages, the area of the cytoplasm and nuclei of labelled hepatocytes were more than those of the unlabelled cells. No significant changes were observed in the nucleoli. The area of the endoplasmic reticulum and mitochondria were less in the labelled hepatocytes than in the unlabelled hepatocytes. The number of the mitochondria was less in the labelled hepatocytes than in the unlabelled hepatocytes. The cell organelles of the hepatocytes that were synthesizing DNA were not well developed, as compared to those not synthesizing DNA during the postnatal development.     

Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.     

Age-related hematological changes in normal F344 rats: during the neonatal period.

In order to clarify age-related changes in hematological values of normal rats after birth, blood samples from neonatal F344 rats of both sexes were examined periodically during the period from 0 to 40 days postpartum. The erythrocyte count (RBC) increased with time after birth as a function of age. In contrast, the reticulocyte count (Retics) continuously decreased with time after birth. Hemoglobin (Hb), hematocrit (Ht), and the mean corpuscular hemoglobin concentration (MCHC) tended to decrease after birth until weaning (about 21 days postpartum), but they began to increase after weaning. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH) also gradually decreased after birth until weaning, but they were unchanged thereafter. The platelet count (PLT) gradually increased after birth and reached a plateau at weaning. Microscopic examination of blood smears revealed that erythrocytes at birth had characteristic morphological features such as anisocytosis, polychromasia, basophilic stippling, Howell-Jolly body, and erythroblastosis. These characteristic features, however, disappeared by 30 days after birth. The total leukocyte count (WBC) gradually increased with time after birth, due to an increase in the number of lymphocytes. The lymphocyte count started to rapidly increase within several days after birth and the increase continued thereafter. Other differential leukocyte counts also showed various characteristic patterns of changes during the neonatal period. There were no apparent differences between males and females in these changes in hematological values.     

Radioautographic study on the localization of an anti-allergic agent, tranilast, in the rat liver

In order to demonstrate the localization associated with metabolism of an anti-allergic agent, Tranilast, in the liver, light microscopic radioautography of the liver was performed. Rats were administrated orally with 3H-Tranilast, and were sacrificed at 15 minutes to 24 hours after the administration. The livers were taken out and fixed, embedded and processed for light microscopic radioautography. 3H-Tranilast was absorbed rapidly, and the radioactivity in the liver increased and decreased within several hours. The number of radioautographic silver grains reached a maximum 3 hours after the administration. From 1 to 6 hours after the administration, the silver grains decreased from the portal area toward the central area. Seventy to 80% of all silver grains on the hepatocytes were retained in the cytoplasms of the hepatocytes at any experimental period. From these results, it was concluded that the localization of radioautographic silver grains was associated with Tranilast uptake of hepatocytes in each hepatic lobular compartment and that the metabolic process from uptake to excretion of Tranilast took part in the hepatocytes in each hepatic lobular compartment.     

BALB/C小鼠肝细胞结构的年龄变化——光镜与电镜的计量形态学研究

作者研究了1月、6月和12月三个不同年龄组的BALB/c小鼠的肝细胞结构。利用计量形态学方法在光镜与电镜两个水平上对肝细胞整体及重要细胞器的年龄变化进行定量分圻,发现在所观察的结构参数中大部分随年龄而发生动力学变化。文中讨论了某些变化的可能意义。     

Ultrastructural and DNA Epifluorescence Observations of the Sperm Cells of Rhododendron-- with Emphasis on Biparental Plastid Inheritance

The mature pollen grains of Rhododendron mucronulatum Turcz. conform to the 2-celled type. Sperm cells differentiated within the pollen tube about 24 hours after germination in vitro and paired together, one of which being linked with the vegetative nucleus, forming a male germ unit (MGU). Abundance of plastids, mitochondria, microtubules and single-membrane-bounded vesicles could be visualized in each sperm cell, however, endoplasmic reticulum and Golgi apparatus were scarce. The electron-dense plastids with normal structure gave ring-like or dumbbell appearance in sections. Mitochondria were smaller and less electron-dense' in contrast to the plastids. DNA epifluorescence technique revealed that the generative and sperm cells contained numerous organelle nuclei (nucleoids). There was no difference in nucleoid number between the two sperm cells in a pollen tube. The results confirmed the possible existance of cytoplasmic inheritance potential of the male gametes of Rhododendron.     

Effects of aging on cholinephosphotransferase activity on guinea pig lung mitochondria and microsomes

It is known that the composition of phospholipids in lung changes with age. The final step in thede novo synthesis of phosphatidylcholine, a major component of lung surfactant, by the CDP-choline pathway, requires the enzyme cholinephosphotransferase (CPT). Even though CPT has earlier been proposed to be located exclusively in the endoplasmic reticulum, we have recently demonstrated its presence also in the mitochondria. We have earlier reported a gestational variation of CPT activity in fetal mitochondria and microsomes. In the present study we examined the subcellular distribution of CPT activity in lung as a function of age. After birth, the microsomal CPT activity continued to increase until adulthood (24 wks of age), thereafter it gradually decreased. On the otherhand, the CPT activity of mitochondria continued to increase with the advancement of age and beyond 72 wks of age, it was approximately 2-fold higher than that of the microsomal fraction.     

THE ULTRASTRUCTURE AND HISTOCHEMISTRY OF A NEMATODE-INDUCED GIANT CELL

The development of giant cells induced by the nematode Meloidogyne in tomato roots has been followed under controlled growth conditions and the ultrastructure and histochemistry of these structures have been examined. Entry of the nematode larvae into the roots took place within 24 hours; giant cell formation started on the 4th day and involved breakdown of the cell walls accompanied by thickening of a surrounding giant cell wall and an increase in density and area of the cytoplasm. The nuclei increased in number by simultaneous mitosis throughout a single giant cell. The peak of cytoplasmic density was reached after moulting and during egg production. The rate of protein synthesis in the giant cell is correlated with the rate of growth of the nematode. The giant cell wall is a thick, irregularly surfaced structure which contains all the normal polysaccharide components of a cell wall. The cytoplasm is rich in protein and RNA and contains mitochondria, proplastids, Golgi bodies, and a dense endoplasmic reticulum. The nuclei are large and irregular in shape and contain large nucleoli and a number of Feulgen-positive bodies scattered irregularly along the nuclear envelope. The nucleolus contains RNA and fat as well as Feulgen-positive granules which are revealed after treatment with ribonuclease. It consists of a dense outer cortex surrounding a much lighter central core and is connected at times with the Feulgen-positive bodies in the nucleus. Speculation is provided on the role of these bodies in cytoplasmic protein synthesis.     

Development of binuclearity and DNA-polyploidization in the growing mouse liver

Summary Suspensions of intact liver cells were prepared from 36 male NMRI mice of different age after perfusion of the liver with ice-cold calcium- and magnesium-free phosphate buffer (CMF). The suspensions of the isolated hepatocytes were smeared on slides, fixed, hydrolized and stained by fluorescent acriflavine-Schiff-Feulgen reaction. The number of nuclei per cell was determined in a phase-contrast microscope. Quantitative fluorescent cytophotometric measurements of nuclear Feulgen-DNA were performed on individual nuclei. At the age of 0.5 month, 55% of the hepatocytes were found to be mononuclear, 45% binuclear. In the animal groups aged 1 month, 1.5 months, 3 months, 6 months and 12 months, the percentage of binuclear hepatocytes remained constant at about 80%. Very few liver cells with 3 or 4 nuclei were detected. Feulgen-DNA-measurements revealed a predominance of 2c and 4c nuclei at ages 1 month and 1.5 months with logarithmic increase of 8c nuclei and a decrease of the 2c nuclei. From 1.5 months on 16c nuclei were found, which never exceeded 8%. When total DNA-ploidy of the hepatocytes was calculated similar kinetics at a higher ploidy level were observed. 2c hepatocytes existed in small percentages at very young ages up to 1.5 months, but were also occasionally found in older animals. With increasing age the number of 16c hepatocytes increased logarithmically with a concomitant decrease of the 4c hepatocytes. The percentage of 8c liver cells remained more or less constant. Few hepatocytes with a 32c total DNA content were found in mice aged 3 months and older. In one-year-old mice the mean DNA-ploidy was calculated to be 5.8c per liver nucleus and 10.0c per whole hepatocyte.Supported by Deutsche Forschungsgemeinschaft, Grant No Bo 395/5     

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.     

Phosphoprotein synthesis and secretion by odontoblasts in rat incisors as revealed by electron microscopic radioautography

The secretory pathway of dentin phosphoproteins in rat incisors was studied by electron microscopic radioautography after the injection of 3H-serine, and the results were compared with those using 3H-proline as a tracer. Five min after injection of 3H-serine, radioactivity was found in the rough endoplasmic reticulum. At 10 min, silver grains were observed over the spherical portions of the cisface of the Golgi apparatus. At 20 min after injection, silver grains were seen over the cylindrical portions of the transface of the Golgi apparatus. The secretory granules showed the strongest reaction from 20 min to 1 hr. At 45 min, a significant labeled band appeared at the mineralization front. At 1 hr, the labeling at the mineralization front began to appear in the mineralized dentin, and after 12 hr this labeled band was located within the mineralized dentin. The pathway of 3H-proline was essentially the same as that of 3H-serine, but 3H-proline moved more slowly than 3H-serine, especially in transit from the rough endoplasmic reticulum to the Golgi apparatus. Secretory granules were heavily labeled from 30 min to 1 hr after injection of 3H-proline; no labeling was found at the mineralization front at 45 min. The labeling seen initially over the predentin was over the mineralized dentin no earlier than 6 hr after injection. The labeling pattern with 3H-serine is closely related to the localization of phosphoproteins, whereas the pattern with 3H-proline reflects the production of collagen rather than of phosphoproteins. The present radioautographic results indicate that dentin phosphoproteins are related to secretory granules and are secreted by odontoblasts at the mineralization front and also that phosphoproteins are involved in the process of mineralization of the circumpulpal dentin.     

Ultrastructural Changes in Maturing Sporangia of Albugo Candida

Maturation of sporangia in Albugo sp. involved considerableinternal differentiation. There was a burst of activity in sporangiasoon after their formation when the numbers of mitochondria,and the amounts of endoplasmic reticulum increased. Perinuclearvesicles and smooth surfaced cisternae differentiated into well-developedgolgi apparatus which remained secretory until completion ofmaturation. The cisternae of rough endoplasmic reticulum arrangedthemselves in parallel stacked arrays. Maturing sporangia hadautophagic vacuoles containing various cell organelles. Nucleardegeneration and mitosis proceeded simultaneously. All activitiesdeclined towards the end of sporangial maturation: Golgi dictyosomesbecame quiescent and numbers of mitochondria and amounts ofendoplasmic reticulum decreased. There was a three-fold increasein the thickness of the sporangial wall during maturation.     

人胎儿肝内甲胎蛋白的成份及其对T.B淋巴细胞发育的影响

本文用免疫组织化学方法,分别在冰冻和石蜡切片上,对24例不同胎龄的胎儿肝比较研究了肝内 AFP~+细胞数量及其与 T,B 淋巴细胞之间的关系。结果发现 AFP 仅分布于肝细胞内,其他细胞阴性。不同胎龄的肝脏,AFP 的染色强度和阳性率不同。17周前的胎肝,AFP~+细胞最多以后逐渐减少。出生前的肝脏内只有少数 AFP~+细胞。AFP~+细胞的多少与 B 细胞分化发育无多大关系,但与 T 细胞似乎关系密切,两者呈负相关,即 AFP~+细胞多时,T 细胞很少,AFP~+减少时,T 细胞增加,提示 AFP 对 T 细胞具有抑制作用。同时也证明 B 细胞在胎肝内受 T 细胞的影响不大,主要依赖于肝脏的微环境。另外对 AFP 的生物学意义也进行了讨论。     

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. I. Observations in hepatocytes

To study the site of incorporation of sialic acid residues into glycoproteins in hepatocytes, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of [3H]N-acetylmannosamine (8 mCi) and then 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. Concurrent biochemical experiments were carried out to test the specificity of injected [3H]N-acetylmannosamine as a precursor for sialic acid residues of glycoproteins. In radioautographs from rats and mice sacrificed 10 min after injection, grain counts showed that over 69% of the silver grains occurred over the Golgi region. The majority of these grains were localized over the trans face of the Golgi stack, as well as over associated secretory vesicles and possibly GERL. In rats, the proportion of grains over the Golgi region decreased with time to 37% at 1 h, 11% at 4 h, and 6% at 24 h. Meanwhile, the proportion of grains over the plasma membrane increased from 4% at 10 min to 29% at 1 h and over 55% at 4 and 24 h; two-thirds of these grains lay over the sinusoidal membrane, and the remainder were equally divided over the lateral and bile canalicular membranes. Many silver grains also appeared over lysosomes at the 4- and 24-h time intervals, accounting for 15-17% of the total. At 3 and 9 d after injection, light microscope radioautographs revealed a grain distribution similar to that seen at 24 h, with a progressive decrease in the intensity of labeling such that by 9 d only a very light reaction remained. Because our biochemical findings indicated that [3H]N-acetylmannosamine is a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids), the interpretation of these results is that sialic acid is incorporated into these molecules in the Golgi apparatus and that the latter then migrate to secretion products, to the plasma membrane, and to lysosomes in a process of continuous renewal. It is possible that some of the label seen in lysosomes at later time intervals may have been derived from the plasma membrane or from material arising outside the cells.