Variability in gold bead density in cells

Summary Variability in gold bead distribution between individual cells was demonstrated in both pituitary melanotrophic cells immunocytochemically reacted for ACTH and in neurohypophysia terminals reacted for oxytocin-neurophysin. Gold beads were confined to the secretory granules compartment of both tissues. Density of gold beads in melanotrophic cells reacted for ACTH varied from 100–480 gold beads/m². A much narrower range of gold beads distribution (460–900 gold beads/m²) was observed in axons of the neurohypophysis reacted with anti-oxytocin-neurophysin. These results indicate that the labeling density varies from cell to cell (as well as axon terminals) within morphologically homogeneous population. Thus, it may reflect certain physiological differences between cells. A suggestion is being made that mean gold bead density coefficient of variation should be calculated by comparison between individual cells.     

Localization of anionic constituents in mast cell granules of brachymorphic (bm/bm) mice by using avidin-conjugated colloidal gold

We used the egg avidin gold complex as a polycationic probe for the localization of negatively charged sites in the secretory granules of mouse mast cells. We compared the binding of this reagent to mast cell granules in wild-type mice and in congenic brachymorphic mice in which mast cell secretory granules contained undersulfated proteoglycans. We localized anionic sites by post-embedding labeling of thin sections of mouse skin and tongue tissues fixed in Karnovsky’s fixative and OsO₄ and embedded in Araldite. Transmission electron microscopy revealed that the mast cell granules of bm/bm mice had a lower optical density than those of wild-type mice (P<0.001) and a lower avidin gold binding density (by approximately 50%, P<0.001). The latter result provided additional evidence that the contents of mast cell granules in bm/bm mice were less highly sulfated than in those of wild-type mice. In both wild-type and bm/bm mast cells, the distribution of granule equivalent volumes was multimodal, but the unit granule volume was approximately 19% lower in bm/bm cells than in wild-type cells (P<0.05). Thus, bm/bm mast cells develop secretory granules that differ from those of wild-type mice in exhibiting a lower optical density and slightly smaller unit granules, however the processes that contribute to granule maturation and granule-granule fusion in mast cells are operative in bm/bm cells.     

Immunolocalization of synexin (annexin VII) in adrenal chromaffin granules and chromaffin cells: evidence for a dynamic role in the secretory process

Summary Synexin (annexin VII) is a Ca²⁺- and phospholid-binding protein which has been proposed to play a role in Ca²⁺-dependent membrane fusion processes. Using a monoclonal antibody against synexin, Mab 10E7, and immunogold, we carried out a semiquantitative localization study of synexin in bovine adrenal medullary chromaffin granules, and in resting and nicotine-stimulated adrenal chromaffin cells. Isolated chromaffin granules contained very little synexin, whereas chromaffin granules aggregated with synexin (24 g/mg) and Ca²⁺ (1 mM) clearly showed synexin-associated immunogold particles in the vicinity of the granule membrane (1.88 gold particles per granule profile). In isolated, cultured adrenal chromaffin cells, synexin was present in the nucleus (5.5 particles/m²) and in the cytosol (5.3 particles/m²), but mainly around the granule membrane in the granular cell area (11.7 particles/m²). During the active phase of cholinergically stimulated catecholamine secretion, the amount of synexin label was reduced by 33% in the nucleus, by 23% in the cytosol, and by 51% in the granule area. The plasma membrane contained a small amount of synexin, which did not significantly change upon stimulation of the cells. We conclude that synexin is involved in the secretory process in chromaffin cells.     

Light- and electron-microscopic radioautographic study of glycoprotein secretion in the granular duct of the submandibular gland of the male mouse

Summary L-³H-fucose was injected intravenously into adult male mice, after which, at different time intervals, the submandibular glands were removed and processed for light-and electron-microscopic radioautography. This radio active hexose was taken up by newly synthesized glycoproteins in the cells lining the granular ducts which were maximally labeled at 4 h after injection. Between 4 and 72 h the amount of labeled glycoproteins decreased moderately indicating that these macromolecules undergo a slow renewal. The main subcellular site of incorporation of 3 H-fucose into glycoproteins was the Golgi apparatus. From this organelle labeled glycoproteins were transferred to small secretory granules (diameter up to 1.0 m) located not only near the Golgi region but also throughout the apical cytoplasm. At 1 h after injection the concentration of label reached a maximum in the small secretory granules and labeling of medium (diameter between 1.1 and 2.0 m) and large (diameter over 2.0 m) granules was very low. At this postinjection interval the secretion product inside the lumen of the duct was already labeled. Between 1 and 72 h after injection the concentration of radioactivity in the small secretory granules decreased intensely while increasing in the medium and in the large ones. The concentration of fucose label reached a maximum in the medium secretory granules at 24 h and in the large ones at 72 h after injection. Additional experiments using mice previously injected with 4 intraperitoneal doses of ³H-fucose given 3 h apart demonstrated that the large granules undergo a very slow renewal. Some were found to be labeled as long as 28 days after administration of ³H-fucose. Recorded in this latter series of experiments was the labeling pattern of dense bodies that were regularly visualized in the cells lining the granular ducts. Their significance in the secretory process is discussed. In conclusion, newly synthesized glycoproteins are transferred from the Golgi apparatus to small secretory granules which carry a readily releasible pool of these macromolecules to the lumen of the duct. The small secretory granules also transfer newly synthesized glycoproteins to medium and large secretion granules which store a pool that is released very slowly. This characterizes the large secretory granules as the intracellular sites of storage of secretion products. The results of this investigation were correlated with the knowledge about the chemical composition of the different macromolecules that are known to be synthesized by the secretory cells of the granular ducts of the submandibular gland of the mouse.     

Changes in the distribution of anionic constituents in secretory granules of mouse pancreatic acinar cells after pilocarpine-induced degranulation.

We used cationized colloidal gold (CCG) to investigate the distribution of anionic sites in different secretory granules of mouse pancreatic acinar cell regranulation. Localization of anionic sites with CCG was carried out on ultrathin sections of a mouse pancreas, fixed in Karnovsky's fixative and OsO4 and embedded in Araldite. After pilocarpine-stimulated degranulation, there was a marked diminution in the anionic charge density of immature and mature granules of the 4-hr group (approximately 43.0 gold particles/microm2) compared to the 8-hr mature granules group (approximately 64.6 gold particles/microm2). Scattergram analysis to investigate the correlation between section profile size and cationized gold labeling density revealed a reverse correlation, the small granule profiles demonstrated a higher density compared to the larger profiles of the same group. On the basis of these observations, it appears that a post-translational processing of secretory content influences the granule anionic charge and thus may affect the intragranular buffer capacity.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Ultrastructural immunocytochemical localization of l-glutamate decarboxylase and GABA in rat pancreatic zymogen granules

Summary The ultrastructural immunohistochemical localization of gamma aminobutyric acid (GABA) and its regulating enzymes, l-glutamate decarboxylase (GAD) and gamma aminobutyrate--ketoglutarate transaminase, was determined utilizing an immunogold post-embedding protocol in pancreatic exocrine tissue. Within the acinar cell, GABA and its biosynthetic enzyme, GAD, were localized in zymogen granules. Quantitative analysis of the GABA immunoreactivity in the acinar cell revealed 1.7±0.5 gold particles/m² over the cytoplasm, 36.6±14.1 gold particles/ m² over the zymogen granules, and 2.9±2.1 gold particles /m² over the mitochondria. Quantitative analysis of the distribution of colloidal gold particles, representing glutamate decarboxylase immunoreactivity in the acinar cells, revealed 38.4±2.5 gold particles/m² over the zymogen granules, 4.7±1.1 gold particles/m² over the mitochondria and 6.3±0.5 gold particles/m² over the remainder of the cytoplasm. Substitution of normal sheep serum for the sheep anti-glutamate decarboxylase serum revealed a significant (p< 0.001) decrease of the colloidal gold particle distribution over the zymogen granules and cytoplasmic compartments of the acini. Gamma aminobutyrate --ketoglutarate transaminase, the catabolic enzyme for GABA, was not detected in the mitochondria, zymogen granules, and cytoplasm of the acinar cell, suggesting that GABA is not catabolized within the acinar cell. Preabsorption and substitution controls resulted in an absence of labeling. These results suggest that GABA may act extracellularly and/or have a role within the zymogen granule in the exocrine pancreas.     

Heparin potentiates in vivo neutrophil migration induced by IL-8

Chemokine IL-8 attracts neutrophils by a haptotactic gradient, made possible by its interaction with proteoglycans of the extracellular matrix. Heparan sulfate, but not heparin, potentiates the attraction exerted in vitro by IL-8. In the present study we first confirmed this in vitro phenomenon, observing that IL-8 activity was potentiated 100% by heparan sulfate, but not by heparin. Then, we evaluated the interference of heparan sulfate or heparin on in vivo neutrophil migration induced by IL-8. The activity of rat IL-8 (3.5 g/animal) preincubated with heparan sulfate (50 g/animal) or heparin (77 g/animal) was assayed on the rat dorsal air pouch. Contrary to in vitro experiments, heparin, but not heparan sulfate, potentiated the in vivo IL-8 activity two-fold. We investigated the relationship between this observation and that reported by others, that IL-8-induced migration depends on the presence of mast cells, which contain heparin-rich granules. We studied the neutrophil migration induced by IL-8 (3.5 g/animal) into the rat peritoneal cavity depleted of mast cells. Neutrophil migration was reduced by 32% when compared to that observed in normal animals. The response of depleted rats was reconstituted by preincubation of IL-8 with heparin (77 g/animal). These data suggest that heparin released from cytoplasmic granules may be the contribution of mast cells to IL-8-induced neutrophil migration.     

Ultrastructural immunolocalization of basic fibroblast growth factor to lipid bodies and secretory granules in human mast cells

Isolated human lung mast cells were used to identify subcellular sites of basic fibroblast growth factor using a postembedding immunogold method. The factor was present in quantity in secretory granules and cytoplasmic lipid bodies. Cisterns of smooth endoplasmic reticulum and ribosome clusters, closely associated with lipid bodies, contained the factor as did the nuclear matrix. Factor-positive lipid bodies were adjacent to nuclear pores and often indented perinuclear cisternae. Altered secretory granules with reduced density, characteristic of secretion by piecemeal degranulation in mast cells, showed reduced gold label for basic fibroblast growth factor; small, electron-lucent (80–100nm) transport vesicles near altered granules were labelled for the factor. Since these mature mast cells do not display extensive arrays of classical secretory organelles, such as rough endoplasmic reticulum and Golgi structures, these new subcellular localizations for basic fibroblast growth factor suggest several possible alternative release routes for a cytokine devoid of a signal sequence characteristic of regulated secretory proteins.     

Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

Summary Enterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 m in length and 1–2 m in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.     

Artemisia annua L.: dedifferentiated and differentiated cultures

Dedifferentiated and differentiated tissue cultures ofArtemisia annua L. for artemisinin production were carried out. The calluses were initiated on MS medium supplemented with sucrose (30 g l^-1), myoinositol (100 mg l^-1) and RT vitamins. The auxins used were naphtaleneacetic acid (NAA), indoleacetic acid (IAA), indolebutyric acid (IBA) and 2,4-dichlorophenoxyacetic acid (2,4-d). These were added to the basal medium either singly or in combination. The best results were obtained with 2.4-d (4.5 M : 0.02 d^-1) and NAA (5.4 M : 0.06 d^-1). Cell suspensions were established on the same media without agar. Suspension cultures showed different morphological characteristics according to the plant growth regulator supplied. Organized cultures were initiated from callus obtained on 2,4-d (4.5 M) and from bud cultures. Medium containing 6-benzylaminepurine (BA) (8.9 M)+NAA (0.54 M); Zeatin (45.62 M)+NAA (5.37 M) or BA (8.9 M) stimulated both organogenesis in callus (frequency of induction =50%) and semi-organized tissue in shoot buds. BA (13.32 M)+NAA (1.08 M) or BA (13.32 M) only stimulated multiple shoot cultures (frequency of induction =80%). Regarding artemisinin content, while the values obtained were 1.13 and 0.78 mg gDW^-1 in primary callus, artemisinin was not detected in cell suspension and only traces of it were found in multiple shoot cultures.     

Evidence for the location of divalent cation binding sites on the chloroplast membrane

Summary We have used a combination of chemical labeling and detergent fractionation techniques to locate the divalent cation binding sites on the chloroplast membrane. We determined the Ca²⁺-binding properties of Triton X-100 subchloroplast particles. Photosystem II (TSFII) particles showed one binding site withn=8.4 moles-mg chl^–1 andk _d =20 m. Photosystem I (TSFI) particles contained two binding sites. The first had ann=1.5 moles-mg chl^–1 andk _d =4 m. The second had ann=9.6 moles-mg chl^–1 andk _d =160 m. We have previously shown (Prochaska & Gross,Biochim. Biophys. Acta 376:126, 1975) that the divalent cation binding sites could be blocked using a water-soluble carbodiimide plus a nucleophile. Chlorophylla fluorescence and lightscattering changes were affected at the same carbodiimide concentrations emphasizing the relationship between these processes. The carbodiimide-sensitive sites were found to be located on the Photosystem II particles. A direct correlation between the inhibition of calcium binding and the carbodiimide-mediated incorporation of a (¹⁴C)-nucleophile was observed upon varying such parameters as carbodiimide concentration, nucleophile concentration, pH, and time of reaction. The presence of CaCl₂ during the carbodiimide plus nucleophile modification procedure decreased the incorporation of (¹⁴C)-nucleophile, emphasizing the competition of the CaCl₂ and the modification reagents for some of the same sites. Sodium dodecylsulfate gel electrophoresis of chlorophyll protein aggregates suggested that the site of competition of the calcium chloride and the modification reagents was the light-harvesting chlorophylla/b protein.     

Mutant vasopressin precursor in the endoplasmic reticulum of the Brattleboro rat. Ultrastructural evidence from individual “vasopressin” cells localized with the light microscope by use of a new gold/silver method for immunostain enhancement

Summary This ultrastructural study demonstrates that the vasopressin immunoreactivity found in the occasional, densely stained cells in the hypothalamus of the homozygous Brattleboro rat is localized in the rough endoplasmic reticulum. 50-m Vibratome sections were stained with anti-vasopressin serum by use of a peroxidase method with 3,3-diaminobenzidine as chromogen. The diaminobenzidine end-product has a specific capability to bind gold particles from a chloroauric acid solution and the bound gold was used to precipitate silver grains from a silver developer. The stained sections were flat embedded in resin and ultrathin sections were cut of areas containing the immuno-identified occasional cells. In these densely stained, vasopressin-immunoreactive cells of homozygous Brattleboro rats the rough endoplasmic reticulum was dilated. The lumen of the reticulum contained both end-products of diaminobenzidine and gold/silver grains, but some parts of the reticulum appeared unstained. No other cell organelles were immunostained and no secretory granules were found. In control rats, gold/silver deposits were found throughout the cytoplasm of vasopressin-immunoreactive cells. In these immunostained cells secretory granules were seen.     

Intragranular processing of pro-opiomelanocortin in the intermediate cells of the rat pituitary glands

Summary Quantitative immunocytochemical studies were done by using the immunogold technique on sections of the intermediate lobe of rat pituitary. Antibodies raised (in rabbits) against the precursor proteins pro-opiomelanocortin (POMC) and ACTH were used. The results clearly indicate that the immature granules are the major site of POMC, as their antigenic density (gold beads/m²) was almost 3 times as high as that of ACTH. In the mature igranules, the antigenic density of ACTH was increased by 2.7-fold compared with the immature granules. Using a computer-assisted method, it was possible to categorize the granules antigenic density according to their size. Using this approach it was found that the antigenic density of POMC remained constant in all mature granules of varied sizes, whereas the antigenic density of ACTH decreased with increasing granule size. The relationship between granule size, degree of maturation, and antigenic density is discussed.     

Localization of the two major allergens in rye-grass pollen using specific monoclonal antibodies and quantitative analysis of immunogold labelling

Summary The intracellular localization of the two major allergens, Lol p I and Lol p IX, in rye-grass anthers was examined using monoclonal antibodies FMCA1 (specific for Lol p I) and FMCA7 (specific for Lol p IX) with immunocytochemical techniques and quantitative analysis. A newly developed anhydrous fixation technique in a mixture of glutaraldehyde, paraformaldehyde and 2, 2-dimethoxypropane followed by embedding in LR Gold resin resulted in both improved infiltration of pollen grains compared with existing techniques and the localization of these water-soluble antigens in their original sites compared with diffusion artefacts following aqueous methods. After anhydrous fixation, Lol p I was predominantly located in the electron-opaque regions of the cytosol of the vegetative cell of the tricellular pollen grains (24 counts m^-2), whereas Lol p IX was detected mainly within starch granules (16 counts m^-2). For both Lol p I and Lol p IX, similar labelling was detected in the cells of the endothecium and middle layer (18 counts m^-2), but none was found in the tapetal cells or orbicules.     

The intracellular pathway of antagglutinin secretion in the boar caput epididymidis as revealed by immunogold labelling

Summary Antagglutinin, a specific protein synthesized by the boar epididymis, was localized by an ultrastructural immunogold-labeling procedure in the principal cells of the three regions of the caput epididymidis, most notably at the sites of synthesis and secretion. The intensity of the reaction was variable in the three epididymal zones. Labeling was of low intensity in the proximal and middle caput, except in the granules of the latter. These granular storage sites did not correspond to typical secretory granules but appeared to be intracellular sites of degradation of this protein. In the distal caput, which was devoid of these granules, intense secretory activity for antagglutinin was detected. Few gold particles were localized in the RER profiles but labeling was detected in the Golgi zone, in numerous dense vesicles, in structures distributed between the Golgi zone and the apex of the cell, and in the epididymal lumen. This study has enabled us to visualize immunocytochemically antagglutinin along its intracellular secretory pathway, i.e. at the site of its synthesis, during its passage via the Golgi zone, and its intracellular transport to the lumen.     

Cell death and the regulation of populations of cells in the periodontal ligament

Summary The contribution of cell death in regulating cellular populations of periodontal ligament was studied in young adult rats. Mandibular first molar periodontium was prepared for light-microscopic radioautography after a pulse of ³H-thymidine in 6 rats and for electron microscopy in 4 rats. The labeling index for ³H-thymidine and the density of fibroblast-like cells were computed from radioautographs. The percentages of dying or dead cells and macrophages were computed from electron micrographs. The labeling index of cells within 20 m of bone and cementum was significantly lower (p<0.01) than the labeling index within the body of the periodontal ligament. The patterns of cellular density and indices of death were the inverse of the labeling indices. Macrophages were plentiful (% macrophages = 3.68%±0.30) and were clustered around blood vessels (mean distance from blood vessel=2.3 m). However, only 10% of dying or dead cells were within 10 m of blood vessels. These data show that death of cells in the periodontal ligament may, in part, balance production of cells by mitosis. The relationships between labeling index, index of death, and cellular density suggest that cells born in the middle of the periodontal ligament may migrate to regions of high cellular density near bone and cementum, and that they may die there. Macrophages do not appear to be associated with dying cells of the periodontal ligament.     

The ultrastructural basis for the identification of cell types in the pancreatic islets I. Guinea pig

Summary The pancreatic islets of the guinea pig have been studied by light and electron microscopy. The B granules in glutaraldehyde-fixed tissue often are cup-shaped with an indentation visible on one side of the granule. Phosphotungstic acid hematoxylin (PTAH) positive cells have been characterized by electron microscopy as three subtypes based on the size of the secretory granules. A_a cells are the most common and have secretory granules around 200 m in diameter. A_b cells have large secretory granules around 300 m in diameter and are relatively infrequent. A_c cells are the least common and have small (160 m) granules. Characteristic D cells are identifiable by electron microscopy and, on the basis of the subsequent study (Munger, Caramia, and Lacy, 1965), are identified as the silver positive cells observed by light microscopy.This investigation was supported in part by United States Public Health Service research grants GM-10102 and GM-03784 from the Institute of General Medical Sciences, and AM-01226 from the Institute of Arthritis and Metabolic Diseases.-The authors wish to acknowledge the valuable technical assistance of Mrs. Aileen Sevier and Mrs. Lidia Donohue.     

An enhanced incorporation of fatty acid into phosphatidyl choline that parallels histamine discharge in mast cells

Summary Purified rat peritoneal and pleural mast cells preincubated briefly with radioactively labeled fatty acid were treated with A23187, which bypasses primary receptors in stimulating exocytosis. An enhanced incorporation of fatty acid into phosphatidyl choline (PC) that occurred in parallel with histamine release at 24–25°C was observed and was initially proportional to the total amount of histamine discharged. Enhanced PC labeling and histamine secretion were also proportional at temperatures ranging from 17–37°C. Both radioactive linoleic and palmitic acids were incorporated selectively at the -position of the glycerol backbone of PC. PC labeling by [³H]choline was not detectably different in control and stimulated cells, and phosphatidic acid did not exhibit selectively enhanced -acylation. Thus, the stimulated labeling in A23187-treated cells may occur secondary to the action of a phospholipase A₂ that favors PC as a substrate.Other peritoneal cell types exhibit a very similar A23187-stimulated selective labeling of PC. Therefore, autoradiography has been used to provide a direct demonstration that in purified preparations, mast cells are the principal cell type engaged in A23187-elicited incorporation of fatty acid into PC. The efficacy of this approach has relied on special procedures devised to obtain significantly different autoradiographic grain densities between control and stimulated preparations that can be attributed to differences in the level of [³H]palmitate-labeled PC.Preliminary tests using compound 48/80 as a secretory stimulus for mast cells have identified a similar selectively enhanced PC labeling. In either case, however, consideration of possible relationships between PC metabolism and the secretory process are premature since they have not been tested directly.