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Glycosylation of apoproteins of rat very low density lipoproteins during transit through the hepatic Golgi apparatus.

The glycosylation of apo very low density lipoproteins (apo-VLDL) in vivo was studied by following the incorporation of [14C]glucosamine into several groups of apoproteins of VLDL isolated from hepatic Golgi fractions and from serum of sucrose-fed, colchicine-treated rats. Simultaneous incorporation of [3H]leucine was used to quantitate the apoproteins following separation by polyacrylamide gel electrophoresis. Experimental conditions were selected so that the 14C:3H ratio in the apoproteins permitted estimations of the extent of glycosylation by glucosamine and its metabolites. A rapidly decreasing 14C:3H ratio was noted in serum apo-VLDL for the first 30 min after administration of the isotopically labelled precursors, followed by stabilization of the ratio. These data are consistent with the glycosylation of a preformed pool of apo-VLDL, probably apo-B. Glucosamine was progressively incorporated into apo-VLDL during transition from the forming face of the Golgi apparatus to the secretory vesicles, as indicated by an increasing 14C:3H ratio. On the other hand, the ratio of the rapidly migrating apoproteins of VLDL, corresponding to the apo-C-II and apo-C-III, showed the opposite trend, as did total apo high density lipoprotein (apo-HDL) and the rapidly migrating bands of apo-HDL. Division of the rapidly migrating apoproteins of VLDL into upper bands (probably apo-C-II and apo-C-III-0) and lower bands (probably apo-C-III-3) resulted in a 14C:3H ratio near zero in the upper band apoproteins, consistent with the absence of carbohydrates. The lower band showed a rising 14C:3H ratio during transition through the Golgi apparatus, suggesting increased glycosylation, The decreasing 14C:3H ratio in the rapidly migrating proteins is therefore due to the acquisition of apo-C-II and apo-C-III-0 by VLDL during passage from the forming face to the secretory vesicles of the Golgi apparatus.

Animals

A fine-structural analysis of mouse molar odontoblast maturation.

The first mandibular molars of the Swiss albino mice, 1 through 4 days of age, were fixed in glutaraldehyde or Karnovsky's fixative. The tissues were postfixed in OSO4, dehydrated and embedded in Epon. The prepolarizing, polarizing and secretory odontoblasts were described. The prepolarizing cells, located in the vicinity of the cervical loop, were mesenchymal-like in morphology. The cells of the polarizing stage possessed organelles indicative of protein synthesis. The nucleus was located proximally. Aperiodic fibers were evident in the wide basement membrane. The secretory odontoblasts were long, slender, polarized cells closely adjoining one another. Each odontoblast possessed six morphologically discernible regions: (1) an infranuclear region, limited in size and containing few cellular organelles; (2) a nuclear region, housing the oval nucleus and a few associated lamellae of rough endoplasmic reticulum as well as a limited number of mitochondria; (3) a supranuclear rough endoplasmic reticulum region, possessing an abundance of these organelles as well as some mitochondria and secretory vesicles; (4) a Golgi region, occupying the middle third of the cell, housing the elements of an extensive Golgi apparatus which was surrounded by peripherally located profiles of rough endoplasmic reticulum; additionally, this region contained smooth endoplasmic reticulum, mitochondria, numerous secretory granules and vesicles and occasional intracellular collagen fibers; (5) an apical rough endoplasmic reticulum region, containing a rough endoplasmic reticulum component that was less extensive than its supranuclear counterpart; in addition, this region was the one richest in mitochondria and contained a plethora of secretory vesicles and granules; (6) the odontoblastic process, a region mostly void of organelles, containing various secretory products, some of which appeared to be in the process of being released extracellularly into the surrounding dentin matrix.

Animals

Association of centrioles with clusters of apical vesicles in mitotic thyroid epithelial cells. Are centrioles involved in directing secretion?

The ultrastructure of thyroid epithelial cells in mitosis has been investigated. A spatial association is described between clusters of apical vesicles (believed to contain thyroglobulin destined for secretion into the follicular lumen) and centrioles, in late prophase and late telophase cells. Quantitative techniques demonstrate the statistical significance of this association and suggest that it is not related to proximity of the Golgi apparatus or to the location of the centriole in the cell, which changes considerably during these phases of mitosis. The physical basis for this association remains uncertain, but microtubules emanating from the pericentriolar area may be involved. In interphase cells, centrioles are located very close to the follicular lumen, where the majority of apical vesicles are also found. The association of centrioles with clusters of apical vesicles also in mitotic cells suggests that in interphase cells the apically located centrioles may serve as a focus for apical vesicles, helping to direct these secretory vesicles toward the follicular lumen and to maintain cellular polarization. Previous studies demonstrating that centrioles can act as microtubule organizing centers in interphase cells and studies linking microtubules and secretion also tend to support this hypothesis.

Animals

Evidence against phospholipid asymmetry in intracellular membranes from liver.

We have studied the distribution of phospholipids across the membrane of microsomal vesicles and Golgi-derived secretory vesicles from rat liver by the use of phospholipases. Model studies on single-bilayer phospholipid vesicles showed that phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) cleaved at least 80% of the lipids on the outer surface of such vesicles without significant attack on the inner surface. In microsomal vesicles approximately 40% of the outer surface phospholipids were cleaved before the enzyme gained access to the interior of the vesicles. The same conclusion was reached for Golgi vesicles. By following the degradation of the three major phospholipids in intact microsomes and in extracted lipids we found that the same fraction of each of these phospholipids was exposed on the outer surface of the microsomal vesicles. Corresponding experiments with Golgi vesicles showed that distinctly different fractions of phosphatidylcholine and phosphatidylethanolamine were present on the surface of these vesicles. However, the difference was accounted for by enrichment of phosphatidylcholine in intravesicular particles rather than by asymmetry across the vesicle membrane. The results from specific hydrolysis of phosphatidylinositol confirmed an essentially symmetric distribution of this phospholipid across the microsomal and the Golgi vesicle membranes.

Animals

Parathyroid hormone biosynthesis. Correlation of conversion of biosynthetic precursors with intracellular protein migration as determined by electron microscope autoradiography.

The formation of parathyroid hormone (PTH) in the parathyroid gland occurs via two successive proteolytic cleavages from larger biosynthetic precursors. The initial product coded for by PTH mRNA is pre-proparathyroid hormone (PreProPTH), a polypeptide of 115 amino acids. Within 1 min of synthesis, the polypeptide, proparathyroid hormone (ProPTH), is formed as a result of the proteolytic removal of the NH2-terminal 25 amino acids from Pre-ProPTH. After a delay of 15-20 min, the NH2-terminal six-amino acid sequence of ProPTH is removed to give PTH of 84 amino acids. To investigate the subcellular sites in the parathyroid cell where the biosynthetic precursors undergo specific proteolytic cleavages, we examined, by electron microscopy autoradiography, the spatiotemporal migration of autoradiographic grains and, by electrophoresis, the kinetics of the disappearance of labeled Pre-ProPTH and the conversion of labeled ProPTH to PTH in bovine parathyroid gland slices incubated with [3H]leucine for 5 min (pulse incubation) followed by incubations with unlabeled leucine for periods up to 85 min (chase incubations). By 5 min, 85% of the autoradiographic grains were confined to the rough endoplasmic reticulum (RER). Autoradiographic grains increased rapidly in number in the Golgi region after 15 min of incubation; from 15 to 30 min they migrated within secretory vesicles still in the Golgi region and then migrated to mature secretory granules outside the Golgi area. Electrophoretic analyses showed that Pre-ProPTH disappeared rapidly (by 5 min) and that conversion of ProPTH to PTH was first detectable at 15 min and was completed by 30 min. At later times of incubation (30-90 min), autoradiographic grains within the secretion glanules migrated to the periphery of the cell and to the plasma membrane, in correlation with the release of PTH first detected by 30 min. We conclude that proteolytic conversion of Pre-ProPTH to ProPTH takes place in the RER and that subsequent conversion of ProPTH to PTH occurs in the Golgi complex.

Animals

The intracellular pathway for parathormone biosynthesis and secretion.

The initial translation product of parathormone messenger RNA--preproparathormone--is larger than parathormone. Two amino terminal peptide segments of the peptide chain are removed sequentially to form the 84-amino acid hormone. The first cleavage, the removal of a 25-amino acid extension from preproparathormone, occurs in the rough endoplasmic reticulum, and results in the formation of proparathormone. This peptide moves via an energy-dependent mechanism to the Golgi region of the cell where a specific converting enzyme cleaves a basic hexapeptide segment yielding parathormone itself. A part of the newly formed hormone then is enclosed within prosecretory vesicles, transported to the cellular membrane and secreted. Another portion is stored in mature secretory vesicles and is subject to subsequent secretion. The moment to moment control of parathormone secretion by calcium resides at the plasma membrane, but a tightly coupled response at the level of intracellular hormone degradation is also necessary in order to control intracellular hormone levels in the face of rapid changes in secretory rate. Three major secretory products are released from the parathyroid under the control of extracellular calcium: (1) parathormone, (2) a large protein--"parathyroid secretory protein"--whose function is unknown, and (3) peptide fragments of parathormone. Secretion of hormonal fragments adds to the population of parathormone immunoreactivity in the blood. These fragments appear to be similar if not identical to those formed by peripheral metabolism of parathormone in the liver and kidney.

Calcium

Alterations in hepatic fine structure after chronic exposure of rats to dexamethasone.

The objective of this study was to determine the effects of chronic dexamethasone (DEX) administration on hepatic ultrastructure and to correlate these changes with plasma lipoprotein levels. Electron microscopic studies were made of hepatocytes from male rats killed 1, 3 and 5 days after DEX (2 mg, twice per day) administration. Three days after treatment plasma lipoprotein levels were highest and hepatocytes contained regions of the cytosome rich in elements of the smooth endoplasmic reticulum (SER). Osmiophilic particles were present in the tubules and vesicles of the SER, in the saccules and vacuoles of the Golgi complex, in secretory vesicles near the cell surface and in the space of Disse. DEX treatments also caused hepatocytes to accumulate tightly packed masses of beta-particles of glycogen in some regions of the cell while other areas displayed dispersed glycogen particles that were associated with the SER. These observations are consistent with the hypothesis that glucocorticoids 1. cause an elevation of plasma lipoprotein levels by increasing hepatic synthesis and secretion of VLDL, which involves the sequential participation of the ER, the Golgi complex and exocytosis of VLDL-containing vacuoles into the space of Disse, and 2. produce a change in the nature of the association of glycogen particles with the SER membranes in response to the physiological state of the animal.

Animals

Effect of prepartum milk removal on quantitative morphology of bovine lactogenesis.

The effect of prepartum milking on bovine mammary histology and milk composition was studied in five Holstein cows. One udder half was milked twice daily starting 10 days before the estimated calving date. On udder half was not milked until 2 days postpartum when all quarters were milked and two tissue samples per quarter were taken for morphological analyses. Lactose, casein, and fat concentrations differed from prepartum-milked to postpartum-milked quarters (.82, -1.44, and 3.29%). Quarters milked prepartum were more developed: mature epithelium 24.8%, immature epithelium - 8.9%, total epithelium 15.6%, stroma -24.8%, and alveolar lumen 8.5%. Most alveolar cells in prepartum-milked halves had prominent rounded basal nuclei, hypertrophied secretory vesicles, and a mixed array of lipid droplets. Cells within alveoli were morphologically similar. Secretory epithelial cells from postpartum-milked glands had irregular randomly located nuclei, indistinct Golgi, variable lipid content, and less cytoplasm. These differences demonstrate the importance of prepartum-product removal on secretory cell development and subsequent milk production.

Animals

B-cells of the synovial membrane. II. Differentiation during development of the synovial cavity in the mouse.

Study of pre- and postnatal development of the metatarsophalangeal joint of the mouse shows that the synovial cavity (SC) forms before any differentiation of the synovial mesenchyme. The primitive cleft results from degradation of a thin vascular mesenchymal layer in direct contact with the chondrogenic layers. Differentiation of the synovial membrane coincides with clarification of the SC (3rd to 6th day of postnatal life). When dilatation of the SC occurs (6th to 8th day), the two intimal cells types (A- and B-cells) are well identified. The B-cells already show typical features at day 6; their content of typical dense secretory vesicles is comparable to that of the adult B-cells at day 13. The specific secretory function of B-cells could be correlated with the particular structure of the intimal interstitial tissue and could account for the origin of some protein(s) of the synovial fluid.

Animals

B-cells of the synovial membrane. I. A comparative ultrastructural study in some mammals.

Comparative ultrastructural study of the B-cells in the intimal layer of the synovial membrane in mouse, rat, rabbit, guinea-pig and man clearly distinguishes these cells from both the histiocytic A-cells (macrophage-like cells) and the fibroblasts. In addition to the marked development of the rough endoplasmic reticulum and Golgi apparatus, typical dense secretory vesicles apparently of Golgi origin are always found in mouse B-cells and frequently in those of the rat. These secretory characteristics clearly relate these cells to glandular cells engaged in polypeptidic secretion. The variations in the other species studied concern only the figuration of secretory material. Thus, the B-cells appear to constitute a category of secretory cells specific to the synovial membrane, but the function of which has yet to be determined.

Animals

The ultrastructure of the sweat glands of the ox, sheep and goat during sweating and recovery.

The ultrastructure of the sweat glands of cattle, sheep and goats was studied before, during, and after, exposure of the animals to controlled warm environments. In cattle, sweating induced little ultrastructural change in the gland, although fluid-filled spaces appeared between the myo- and secretory epithelial layers. The mechanism appears to be one of fluid transport and exocytosis of secretory vesicles, which in this species seem to be derived from the Golgi apparatus and/or mitochondria. The glands of the sheep and goat also displayed signs of vesicle exocytosis and of fluid transport during sweating. The sweating 'fatigue' in these species was apparently due to failure of the secretory cells, some of which ruptured and were extruded into the lumen. The evidence during subsequent recovery indicates that neighbouring cells spread to make contact, encase remnants of atretic cells between them and the underlying myoepithelium, and engulf them. Sweat in these species appears to be formed (a) by secretion and (b) from cells which can no longer meet the demands of stimulation. The role in sweating of cell replacement, and of undifferentiated cells found between the myo- and secretory epithelia, is discussed.

Animals

A freeze-fracture study of exocytosis and reflexive gap junctions in human ovarian decidual cells.

Fine-structural features of ovarian decidual cells and their mode of secretion were examined by means of freeze-fracture microscopy. Unique cortical peduncular processes contained secretory vesicles within the expanded peduncle tip, the membrane-leaflets of which exhibited a particle-poor E face adjacent to the vesicle lumen and a P face containing a greater particle number. Exocytosis from attached peduncles involved release of vesicular profiles 40-55 nm in diameter; small particles 8.5-11.5 nm in diameter were also observed at degranulation sites. In fractures revealing the E face of the plasmalemma, cytoplasmic portals at the bases of peduncular stalks were distinguishable from endocytic vesicles. The frequent occurrence of reflexive gap junctions associated with peduncles was shown by freeze-fracture. However, there appeared to be no consistent spatial relationship between gap junctions, secretory peduncles, or sites of exocytosis. Freeze-fracture analysis of the topography of reflexive gap junctional profiles revealed that such gap junctions share basic similarities with intercellular gap jum particle-free aisles. The finding in the present study of reflexive gap junctions occurring between peduncles and the cell soma, as well as between peduncles, suggests that the original definitiof the same cell should be broadened to include any gap junctional specialization formed between portions of the plasma membrane of one cell.

Cell Membrane

The pineal gland of the gerbil, Meriones unguiculatus. III. Morphometric analysis and fluorescence histochemistry in the intact and sympathetically denervated pineal gland.

Morphometric analytical procedures were employed to study the pineal gland of the Mongolian gerbil following superior cervical ganglionectomy (SCGX). The purpose of this study was to define the effects of sympathetic denervation on the morphology of the gland at two time periods, 0500 and 1900 h (one hour before lights-on and lights-off, respectively). Fluorescence histochemistry was employed to determine catecholamine and indoleamine content in intact and denervated pineal glands. After SCGX, the pinealocytes decrease in size, concretions are prevented from forming, and the yellow fluorescence in the gland is lost. Following denervation a depression in the volume of most of the pinealocyte organelles, i.e., SER, RER/ribosomes, free cytoplasm, mitochondria and presumptive secretory vesicles, was also observed. However, synaptic ribbons increased in volume in the gerbils that had been killed at 1900 h. It appears that the sympathetic innervation to the pineal gland is a requirement for the presumptive secretory activity of the pinealocytes.

Animals

X-ray microanalysis of calcium binding sites in Paramecium with special reference to exocytosis.

In Paramecium cells Ca++-stimulated triggering of the exocytosis of secretory vesicles ("trichocysts") was achieved by ionophores X-537 A or A 23187. Under triggering conditions electron dense deposits were present in some "resting" trichocysts and regularly in discharging trichocysts; upon subsequent fixation deposits occurred on the trichocyst membrane (on the inner side or within the membrane) and on the "inner lamellar sheath" from where deposits seemed to "radiate" into the secretory materials. Similar results were obtained with glutardialdehyde fixation alone which also triggers exocytosis but only at low concentrations. Element analysis by energy dispersive x-ray microanalysis ascertained the presence of Ca and P in deposits occurring in trichocysts. Those "resting" trichocysts which were devoid of deposits did not contain Ca or P enriched. Hence, an abrupt Ca++-influx into individual trichocysts just before exocytosis seems to be involved in the triggering mechanism, possible in combination with the sudden activation of an ATPase system localized at those sites of the trichocysts which primarily contain the deposits. When paramecia were treated only with Ca++ and then fixed with OsO4 plus oxalate or merely with glutardialdehyde, electron scattering deposits were formed also on the inner side of the cell membrane and within the ciliary shaft (but rarely in trichocysts). Deposits obtained on cilia (including "ciliary granule plaques") also contained Ca, P and S. Cells contain osmiophilic "calcium-storing vacuoles" which were selectively rich in Ca and S but devoid of P.

Animals

Ammonia inhibits protein secretion in isolated rat hepatocytes.

The general secretion of proteins by isolated rat hepatocytes in suspension is inhibited by colchicine, anoxia, or ammonia (NH4Cl). The inhibition by ammonia is accompanied by the cytoplasmic retention and swelling of protein secretory vesicles, suggesting that ammonia accumulates in the vesicles and thereby prevents their translocation to the cell periphery. General protein synthesis appears to be relatively unaffected by ammonia.

Ammonia

Cytochemical study on the mechanism of secretion of catecholamines.

Adrenal medulla and carotid body of cats are studied with acrylic aldehyde-dichromate and glutaraldehyde-dichromate methods for catecholamines. The frequency of dark vesicles and the mean diameter of the cores obtained by these specific methods are compared with those found by the standard glutaraldehyde-osmium method in normal and reserpine-treated animals. In the samples of normal organs fixed with aldehyde-dichromate methods, the number of dark-cored vesicles is significantly higher than in the controls. On the contrary, in the adrenal gland of reserpine-treated specimens the specific methods demonstrate reductions in the frequency of dark vesicles that can be semiquantitatively compared with the known diminution in the content of catecholamines produced by the drug. In the carotid body it was not found a so strict relation, nevertheless the specific methods give a consistently lower frequency of dark vesicles in reserpine-treated than in control animals. It is suggested that the discrepancy between standard and specific methods in the carotid body is due to substances existent in the dark cores contrasted by the former procedure but not by the latter. The constancy of the mean diameter of the cores even in glands that have lost 93% of their granules confirms the suggestion of an all-or-nothing exocytotic mechanism of discharge of the secretory vesicles. The inverse relation shown between the frequency of dark and clear vesicles indicates that membranes emerge in the cytoplasm after exocytosis as clear vesicles.

Adrenal Medulla

Embryologic origin of various epithelial cell types in the thyroid gland of the rat.

Ventral pharyngeal outpocketings and ultimobranchial outpocketings from the 14-day-old fetus of the Fischer rat were isolated before they fused to form thyroid glands. The outpocketings were implanted into different kidney capsules of adult male Fischer rats, and were allowed to grow for several months. Transplants were then excised and examined by electron microscopy. The ultimobranchial outpocketing gave rise to two types of follicles. One contained ciliated cells, cells with an abundant agranular reticulum, U cells with basal hemidesmosomes, and two types of cells with secretory vesicles. The other contained C cells separated from the follicular lumen by a single flat fiber-containing cell. The ventral outpocketing formed typical thyroid epithelium making up the usual thyroid follicles differing from follicles in the thyroid in situ by the absence of C cells. These follicles were functional as determined by autoradiographic studies with 125I but differed from thyroid follicles in situ with respect to size distribution. The results suggest that (1) in the adult thyroid gland the C cell in the usual follicle is an ultimobranchial contribution and (2) the so-called ultimobranchial follicle is ultimobranchial in origin but that the typical thyroid epithelium in mixed follicles of U cells and typical thyroid epithelium is a ventral contribution. The reason for the absence in the thyroid gland in situ of the variety of other cell types observed in ultimobranchial transplants is unknown.

Animals