MICROBODIES IN EXPERIMENTALLY ALTERED CELLS

A rapid and sustained increase in the number of microbodies in liver and kidney cells can be induced in male rats by ethyl chlorophenoxyisobutyrate (CPIB), a hypolipidemic drug. This phenomenon permits investigation of several aspects of microbody behavior in experimental conditions. Reversal experiments demonstrate that liver cells revert to normal between 2 and 3 weeks after withdrawal of CPIB and that one of the mechanisms for removal of excess microbodies is their incorporation into structures indistinguishable from lysosomes. In a state of rapid cell division, such as that present during liver regeneration, microbody proliferation apparently occupies a high biological priority. In necrotic or degenerating cells microbody structure remains relatively normal. The increase in microbodies induced by CPIB is inhibited by chloramphenicol. No increase in microbodies occurred in female rats or in chickens, guinea pigs, or rabbits at the dosage used (0.25% in diet). No changes in microbodies were seen in monkey liver. Catalase activity was generally parallel to the numerical response in microbodies. Additional observations suggest that the microbody response to CPIB is not related to hepatomegaly induced by this agent but may be related to the hypolipidemic effect of CPIB, though hypolipidemia per se is not a specific or sufficient cause of microbody proliferation.     

STUDIES ON THE ENDOPLASMIC RETICULUM : II. SIMPLE DISPOSITIONS IN CELLS IN SITU

A survey of a large number of different cell types has indicated the presence of a network of membrane-bound cavities (the endoplasmic reticulum) in the cytoplasm of all cell types examined, with the exception of the mature erythrocyte. In its simplest form, encountered in seminal epithelia and in leucocytes, the reticulum consists mainly of interconnected strings of vesicles and appears to be randomly disposed in three dimensions. Local differentiations occur within the endoplasmic reticulum of all the cell types studied. The membrane limiting the cavities of the endoplasmic reticulum appears to be continuous with the cell membrane and the nuclear membranes.     

OVARIAN STEROID CELLS : II. The Lutein Cell

The lutein cells of the rabbit exhibit fine structural variations during their life-span of 28 to 30 days. A systematic examination of the corpus luteum reveals that cellular distinctions may be recognized during the first, second, and third stages of pregnancy. The agranular endoplasmic reticulum reveals vesicular, tubular, and cisternal profiles after fixation with each of the following fixatives: glutaraldehyde, osmium tetroxide, and permanganate. The osmolality of the fixing solutions was varied with sucrose and recorded with an osmometer in order to determine the effect of osmotic concentration on the intracellular membranous profiles. It was determined that vesicles and short, branched tubules of similar structure are present in the agranular reticulum when the osmolalities are 300 to 800 milliosmols (iso-osmotic considered 300 milliosmols). At 900 milliosmols, the vesicular or tubular lumen is obliterated. Intracellular membrane profiles do not exhibit interconversions due to hyperosmotic fixative solutions. The agranular endoplasmic reticulum is randomly distributed as short tubular profiles during the first third of pregnancy. A continuity between these membranes and irregular, electron-opaque lipid masses is evident. When physiological and histochemical data indicate that the lutein cell may be storing sterol precursors, cytological observations show that the agranular endoplasmic reticulum exists in a more organized pattern within the cytoplasmic matrix. Vesicular and short tubular, circular aggregations as well as whorled cisternal patterns surround the larger, less electron-opaque lipid droplets. Surface views of cisternal agranular endoplasmic reticulum exhibit tubular extensions, accentuating the continuity between these two profiles. During the progress of pregnancy, the lutein cell increases in diameter, and accumulates both lipid inclusions and aggregations of intracellular membranes. The agranular endoplasmic reticulum may be peripherally packed and arranged parallel to the cell surface during later stages. In the postpartum, degenerating lutein cell, large myelin figures are present which form from the agranular endoplasmic reticulum. These cellular events are discussed in relation to lutein cell activity, including both secretion of product and storage of precursors.     

BRAIN MITOCHONDRIA : II. The Relationship of Brain Mitochondria to Glycolysis

A mitochondrial fraction prepared from calf brain cortex possessed negligible glycolytic activity in the absence of the enzymes of the high speed supernatant fraction. When mitochondria were added to a supernatant system supplemented with optimal amounts of crystalline hexokinase, a 20 per cent stimulation of glycolysis was observed. The supernatant fraction produced minimal amounts of lactate in the absence of exogenous hexokinase; the addition of mitochondria doubled the lactate production. The substitution of glycolytic intermediates for glucose as substrates as well as the addition of exogenous glycolytic enzymes to the supernatant fraction or supernatant fraction plus mitochondria indicated that the mitochondria contributed mainly hexokinase and phosphofructokinase. By direct assay of all of the enzymes of the glycolytic pathway, only hexokinase and phosphofructokinase were shown to be concentrated in the mitochondrial fraction. All other glycolytic enzymes were found to exhibit higher total and specific activities in the supernatant fraction.     

Synthesis of Nitrate Reductase in Chlorella: II. EVIDENCE FOR SYNTHESIS IN AMMONIA-GROWN CELLS

Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti-nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein.     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : II. Ultrastructural Studies

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.     

RNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Increase in Rate of RNA Synthesis during G1

Cultures of mitotic Chinese hamster cells, prepared by mechanical selection, were pulse-labeled with methionine-methyl-¹⁴C or with uridine-³H at different stages in the life cycle. The rate of ¹⁴C incorporation into 18S RNA was measured, as was the rate of uridine-³H incorporation into total RNA for both monolayer and suspension cultures. The rate of incorporation increased continuously throughout interphase in a fashion inconsistent with a gene-dosage effect upon RNA synthesis.     

RNA METABOLISM IN HELA CELLS AT REDUCED TEMPERATURE : II. Steps in the Processing of Transfer RNA

Incubation of HeLa cells at 24°C results in the modification of the processing of pre-tRNA to tRNA. Both methylation and size reduction were shown to take place in vitro when purified pre-tRNA was subjected to processing in a cytoplasmic extract of HeLa cells The migration of pre-tRNA from the nucleus to the cytoplasm was not significantly altered at 24°C     

PHASE CINEMICROGRAPHIC OBSERVATIONS ON CULTURED CELLS. II. MASS MOVEMENT OF CYTOPLASM IN EUPHORBIA MARGINATA

Mass movement is a form of streaming in which distinct quantities of cytoplasm flow as entities along a transvacuolar strand or cytoplasmic striations of the peripheral cytoplasm. An individual mass can move at variable velocities during a brief period of time or change its direction of flow. Two masses, when moving at different velocities in the same or different directions along a strand, can be observed to collide. This can occur repeatedly, resulting in the formation of a mass of considerable size. Many organelles can be observed to move at velocities differing from that of the mass; some can be observed to change directions during their movement. A mass may represent a dilation of one or more microstreams within the cytoplasm. Folding of the microstreams within a mass may explain the changes in the direction of movement observed for some organelles. Several levels of movement are associated with streaming, including those of the ground plasm, of the organelles, of the transvacuolar strands and of the cytoplasm masses. These, and possibly more subtle aspects of the streaming phenomenon, must be incorporated into any theory of streaming.     

The Ultrastructure of the Digestive Glands in Pinguicula vulgaris L. (Lentibulariaceae) Relative to their Function. II. The Changes on Stimulation

The secretory cells of the digestive glands remain highly activeduring the entire period of prey digestion and absorption ofnutrients. They appear to play a major role in gland activity.A model of the digestive gland's activity on stimulation isproposed. It is very similar to that suggested earlier for Dionaeamuscipula. After the digestion and absorption cycle, destructiveprocesses are initiated in the glands. These appear similarto those observed in the glands of the ageing, unstimulatedleaf and are not associated with feeding. Pinguicula vulgaris L. carnivorous plant, digestive glands, ultrastructure, protein secretion absorption, senescence     

Endocrine effects of chronic administration of psychoactive drugs to prepubertal male rats. II. LSD.

The endocrine effects of chronic D-lysergic acid diethylamide (LSD) administration to prepubertal animals were studied by injecting intraperitoneally three times a week for a month either 100 mug or 500 mug of the psychoactive drug per kilogram or the vehicle to groups of Sprague-Dawley male rats starting at 21 days of age. Animals injected with either dosage of LSD had smaller body weights than controls and tail length was significantly reduced in the high dosage group, plasma levels of growth hormone (GH) were decreased in the high dosage group, and pituitary levels in the low dosage group. Plasma levels and pituitary concentrations of luteinizing hormone and follicle stimulating hormone were not significantly modified by the drug. The low dosage of LSD decreased the brain levels of noradrenaline and increased those of dopamine, while the high dosage decreased those of 5-hydroxyindoleacetic acid. These data suggest that LSD, when administered chronically to developing animals, can inhibit body growth probably by altering the secretion of GH through modifications of its neuroendocrine control.     

THE COMPOSITION OF THE DEVELOPING PRIMARY WALL IN ONION ROOT TIP CELLS. II. CYTOCHEMICAL LOCALIZATION

Jensen , William A. (U. California, Berkeley.) The composition of the developing primary wall in onion root tip cells. II. Cytochemical localization. Amer. Jour. Bot. 47(4) : 287—295. Illus. 1960.–The composition of the developing cell wall in the first 2 mm. of the onion root tip was studied using a cytochemical technique that permitted the detection of hemicellulose and the noncellulosic polysaccharides as well as the pectic substances and cellulose. The technique is based on the combination of a differential extraction procedure with the periodic acid-Schiff reaction for carbohydrates. The data obtained indicate that the cells of the apical initials are low in all wall substances but that all of the wall materials are present to some extent. Early in cell development, differences appear in the composition of the walls of the various tissues. The cortical cells are relatively high in the noncellulosic polysaccharides and cellulose while relatively low in the pectic substances and hemicellulose. Very early in development the protoderm is similar to the cortex, but differences develop during the radial enlargement of the cells. During this stage the walls of the protodermal cells are low in the noncellulosic polysaccharides and cellulose and high in pectic substances and hemicellulose. As elongation progresses, these differences are lost and the 2 tissues become very similar. The vascular cell walls are low in the noncellulosic polysaccharides and cellulose and are high in pectic substances and hemicellulose early in development. Later, hemicellulose becomes relatively more important. When the cell wall materials are sequentially extracted, no change in the general morphology of the cell occurs until only the noncellulosic polysaccharides and the cellulose remained. When the noncellulosic polysaccharides are then removed, the cells remain intact but are 30% less in diameter. This suggests that while cellulose is of critical importance, the noncellulosic polysaccharides may play a major role in determining the physical characteristics of the wall.     

ELECTRON MICROSCOPY OF ORAL CELLS IN VITRO : II. Subsurface and Intracytoplasmic Confronting Cisternae in Strain KB Cells

Two special areas involving membranous components in strain KB cells were studied by electron microscopy. The first area described is that of the subsurface regions of two apposing cells in which flattened cisternae (one cisternae in each subsurface region) with membranes spaced 110–230 A apart were found in a confrontation alignment. The long dimension of the profiles of these cisternae ranges from 0.5 to 2 µ. At these intercellular contact areas, each cisterna is closely applied to the adjacent plasma membrane; the intervening space is 60–100 A. We have named the cisternae in these roughly symmetrical areas of cell contact the subsurface confronting cisternae. Communications between these cisternae and those of the rough-surfaced endoplasmic reticulum also were observed. The second area described is that of the intracytoplasmic confronting cisternae. These cisternae were observed as oval or round images about 0.3–1.4 µ in diameter, each image being composed of a pair of concentrically arranged confronting cisternae with membranes spaced 200–400 A apart. The apposing membranes of the two confronting cisternae are electron opaque, smooth, and free of ribosomes, whereas the unapposed membranes are less dense, scalloped, and associated with ribosomes. The spacing between the two intracytoplasmic confronting cisternae is 70–110 A.