Small Secretory Vesicles Research Articles

Rapid Mobilization of Intracellularly Stored RANTES in Response to Interferon-γ in Human Eosinophils

The CC chemokine RANTES is synthesized, stored, and upregulated in response to interferon-gamma (IFN-gamma) in human peripheral blood eosinophils. In this report, we propose that RANTES is rapidly mobilized from eosinophil crystalloid granules during agonist-induced degranulation. We stimulated purified eosinophils (>99%) from atopic asthmatics with 500 U/mL IFN-gamma to analyze the kinetics of mobilization and release of RANTES (0 to 240 minutes). We used subcellular fractionation, immunogold analysis, two-color confocal laser scanning microscopy (CLSM), and enzyme-linked immunosorbent assay (ELISA) to trace the movement of eosinophil-derived RANTES from intracellular stores to release. RANTES was rapidly mobilized (10 minutes) and released after 120 minutes of stimulation (80 +/- 15 pg/mL per 2 x 10(6) cells). RANTES appeared to be stored in at least two intracellular compartments: the matrix of crystalloid granules, detected by major basic protein and eosinophil peroxidase activities, and a specialized small secretory vesicle present in light membrane fractions. The extragranular RANTES was mobilized more rapidly than that of crystalloid granules during IFN-gamma stimulation. This effect was not observed in eosinophils treated with IFN-alpha, interleukin-3 (IL-3), IL-5, granulocyte-macrophage colony-stimulating factor (GM-CSF), or genistein followed by IFN-gamma. Our findings suggest that RANTES may be mobilized and released by piecemeal degranulation upon stimulation, involving transport through a putative pool of small secretory vesicles.

Chitinase secretion by encysting Entamoeba invadens and transfected Entamoeba histolytica trophozoites: localization of secretory vesicles, endoplasmic reticulum, and Golgi apparatus.

Entamoeba histolytica, the protozoan parasite that phagocytoses bacteria and host cells, has a vesicle/vacuole-filled cytosol like that of macrophages. In contrast, the infectious cyst form has four nuclei and a chitin wall. Here, anti-chitinase antibodies identified hundreds of small secretory vesicles in encysting E. invadens parasites and in E. histolytica trophozoites overexpressing chitinase under an actin gene promoter. Abundant small secretory vesicles were also identified with antibodies to the surface antigen Ariel and with a fluorescent substrate of cysteine proteinases. Removal of an N-terminal signal sequence directed chitinase to the cytosol. Addition of a C-terminal KDEL peptide, identified on amebic BiP, retained chitinase in a putative endoplasmic reticulum, which was composed of a few vesicles of mixed sizes. A putative Golgi apparatus, which was Brefeldin A sensitive and composed of a few large, perinuclear vesicles, was identified with antibodies to ADP-ribosylating factor and to epsilon-COP. We conclude that the amebic secretory pathway is similar to those of other eukaryotic cells, even if its appearance is somewhat different.

Multiple and diverse forms of regulated exocytosis in wild-type and defective PC12 cells.

Regulated exocytosis triggered by the photolysis of a caged Ca2+ compound, DM-nitrophen, was investigated by patch-clamp capacitance measurements in two clones of PC12, the first wild-type and the second (PC12-27) defective of both types of classical secretory vesicles together with the neuronal-type receptors for the attachment proteins of the N-ethylmaleimide-sensitive fusion protein, the so called SNAREs. Moreover, the electrophysiological data were correlated with the ultrastructure of resting quick-frozen-freeze-dried cells of the two clones. Wild-type PC12 exhibited two-component capacitance responses, time constants of 30-100 ms and >10 s, that previous studies had suggested to reflect primarily the fusion of the small and large secretory vesicles, each contributing cell surface increases of approximately 10%. Both of these components were largely and specifically inhibited whether cells previously were microinjected with tetanus toxin light chain. In the defective clone, large responses also were recorded ( approximately 19% surface expansion; time constant, approximately 1 s) that, in contrast to those of the wild-type, were entirely resistant to the toxin. Although secretory organelles, i.e., large vesicles and also profiles of small vesicles, were abundant at the cell periphery and often docked to the plasmalemma of resting wild-type PC12, in the defective clone, no superficial accumulation of vesicles was observed. Our coordinate structural and functional results have revealed diversities between the two classical forms of regulated secretion in wild-type PC12 and have provided evidence of a toxin-insensitive form of Ca2+-induced exocytosis, prominent in the defective clone, that may play an important role(s) in cellular physiology.

Analysis of synaptotagmin I–IV messenger RNA expression and developmental regulation in the rat hypothalamus and pituitary

Synaptotagmins are a large family of synaptic vesicle membrane proteins, that appear to be involved in neurotransmitter secretion from small secretory vesicles. We have quantitatively analysed the messenger RNA levels of synaptotagmin I–IV isoforms in adult hypothalamic and pituitary tissues in order to determine which of these isoforms dominate in these tissues which mainly secrete peptides from large dense core vesicles. We also studied the expression of these isoforms during prenatal (E15, and E17) and postnatal (P1, P7, P14 and P21) rat hypothalamic development. In order to assay small individual samples (e.g., pituitary and embryonic tissues), we employed quantitative reverse transcription–polymerase chain reaction methods. Our results show that synaptotagmin I messenger RNA is the most abundant isoform in all tissues, and is about 5.4- or 38-fold higher in hypothalamus than in neurointermediate and anterior pituitary lobe, respectively. Synaptotagmin II, which is very abundant in cerebellum, is relatively low in hypothalamus (5% of cerebellum) and virtually absent from the pituitary. Synaptotagmin III is about 10 times greater in the neural tissues versus the pituitary, and synaptotagmin IV was the least abundant isoform in all the tissues. Developmental analyses of the synaptotagmin isoforms in rat hypothalamus shows that all isoforms are at low levels during embryonic stages and increase postnatally. Synaptotagmin I and II have similar patterns and rise to maximum (adult) levels around P14, whereas synaptotagmin III and IV reach their maximum levels considerably earlier, at P1. These data show that synaptotagmin I is the dominant isoform in both predominantly peptide secreting systems (e.g., in pituitary tissues) and in neurotransmitter secreting systems (e.g., in cerebellum). While the developmental expression patterns of synaptotagmin I and II parallels the temporal development of synaptogenesis in the nervous system, the early maximal expression of synaptotagmin III and IV suggests that these isoforms may have other functions during early postnatal development.

Intracellular trafficking of tropoelastin.

Elastin is secreted as soluble tropoelastin monomers which are then cross-linked in the presence of extracellular microfibrils to form insoluble elastic fibers. Although the secretion of tropoelastin is thought to be mediated and targeted by an intracellular chaperone complex, the intracellular route taken by this protein and the role of such a chaperone complex remain undefined. In the present study, the specific pathway of tropoelastin secretion was investigated in fetal bovine chondrocytes and ligamentum nuchae fibroblasts by immunofluorescence staining and immunoprecipitation of tropoelastin following treatment with secretion-disrupting agents. In untreated cells, tropoelastin is secreted in approximately 30 min. In both cell types, brefeldin A and monensin inhibited secretion of tropoelastin and caused an intracellular accumulation of the protein in the fused ER/Golgi compartment or in the Golgi stacks, respectively. Incubations of longer than 1 h in the presence of brefeldin A result in eventual degradation of tropoelastin in the ER/Golgi compartment (Davis and Mecham, 1996). In contrast, the tropoelastin trapped in the Golgi as a result of monensin treatment steadily accumulated. Agents that elevate intracellular pH, such as ammonium chloride and chloroquine, also caused an intracellular accumulation of tropoelastin which appeared by immunofluorescence staining to be localized in secretory vesicles and/or endosomes. Since weak bases and ionophores alter the morphology of vacuolar compartments, the effect of bafilomycin A1 on tropoelastin secretion was also investigated. This vacuolar H+-ATPase inhibitor prevents acidification of the trans-Golgi network and endosomal compartments without disrupting intracellular organelle formation. When the elastogenic cells were treated with bafilomycin A1, tropoelastin secretion was diminished and an intracellular accumulation of tropoelastin was detected in the trans-Golgi network and small secretory vesicles. These results suggest that tropoelastin may be diverted from the constitutive pathway after exiting the Golgi and instead targeted to an acidic compartment prior to transport to the cell surface. The identity and role of such a compartment in the sorting and/or trafficking of tropoelastin has yet to be determined.

Intracellular transport of basement membrane-type heparan sulphate proteoglycan in adenoid cystic carcinoma cells of salivary gland origin: an immunoelectron microscopic study.

ACC3, a human adenoid cystic carcinoma cell system of salivary gland origin, is able to synthesize and secrete a large amount of basement membrane molecules in vitro. To define the ultrastructural secreting pathway of these molecules, we immunolocalized heparan sulphate proteoglycan (HSPG) in ACC3 for 7 days of culture. In the early stage of culture, the main compartments immunolabelled were rough endoplasmic reticulum (rER) and small secretory vesicles. From days 3 to 4 after plating, it was noticed that HSPG was localized in partially dilated spaces of the perinuclear, rER and Golgi cisternae and in lysosomes or those fused with multivesicular bodies and endosomes. On and after day 5, almost every Golgi apparatus showed marked dilatation of the cisternae and HSPG was immunolocalized in these dilated spaces. In the later stage of culture, autophagic vacuoles or secondary lysosomes, which were simultaneously labelled for HSPG and cathepsin D, were accumulated in the cytoplasm. HSPG deposition in the intercellular space was clearly demonstrated from day 1 and increased during the culture. The results indicate that ACC3 cells have an enhanced turnover cycle for HSPG: not only its biosynthesis but also degradation of both endogenous or exogenous HSPG. Such intracellular events may be reflected in the characteristic histology and biological behaviour of adenoid cystic carcinomas.

Subcellular distribution of 220 kDa antigen in the intercellular spaces of normal human epidermis.

We generated a monoclonal antibody, 7C1, to an extract of pig snout skin used as antigen. The antigen for 7C1 was identified by immunoblotting as a 220 kDa epidermal protein. It was found immunologically to be distributed mainly in the intercellular spaces throughout the living cell layers of normal human epidermis, but its distribution changed in some keratinizing disorders. Study of the association of this protein with subcellular structures by immunoelectron microscopy of normal human skin revealed that it was localized in (1) lamellar granules, (2) intercellular spaces, especially in the lamellar structures derived from the lamellar granules, (3) small vesicles near the cell boundaries and (4) small vesicles in the Golgi apparatus. These results suggest that in humans the 220 kDa protein originates in the Golgi apparatus and is secreted via either the lamellar granules or the small secretory vesicles into the intercellular spaces of the epidermis, where it may be involved in the epidermal permeability barrier function and possibly in keratinizing differentiation.

Exocytotic Competence and Intergranular Fusion in Cord Blood-Derived Eosinophils During Differentiation

AbstractWe studied degranulation of single cord blood-derived mononuclear cells differentiating to eosinophils in cultures containing recombinant human interleukin-5 (rhIL-5) and rhIL-3 by whole-cell patch-clamp capacitance measurements. As in mature cells, degranulation can be stimulated by intracellular application of guanosine-5′-O-(3-thiotriphosphate) (GTP) γS after 10 days in culture, simultaneously with the first morphological appearance of granules. These results demonstrate that the fusion machineries for exocytotic fusion are present and functional as soon as the granules are formed, presumably at the myeloblast stage. In the third week, the total amount of granules exocytosed upon stimulation is similar to that in mature eosinophils from peripheral blood. The capacitance step size distributions in promyelocytes and myelocytes confirm that mature large specific granules are formed by homotypic fusion of unit granules with similar size. Homotypic fusion is facilitated during early stages of differentiation associated with granulogenesis. Between day 10 and day 35 in culture the plasma membrane area of resting cells decreases from ≈700 μm2 to ≈400 μm2, approaching the value of mature cells from peripheral blood. The most prominent decrease occurs between day 25 and day 35 and is accompanied by the appearance of an exocytotic component due to small vesicles. This suggests that a class of small secretory vesicles is formed by endocytosis during a late phase in maturation.

Electron microscopic immunolocalization of the 18 and 29 kilodalton secretory proteins in the mouse epididymis: evidence for differential uptake by clear cells.

In previous studies we reported the synthesis, secretion, and immunolocalization at the light microscopic level of two mouse epididymal proteins, MEP 7 and MEP 10 [Rankin et al. (1992b), Biol. Reprod., 46:747-766]. MEP 7 is the mouse homologue of the rat metalloproteins, AEG/D and E, and MEP 10 is the mouse homologue of the rat retinoic acid binding proteins, B and C. We now describe the immunolocalization of MEP 7 and MEP 10 in the mouse epididymis at the electron microscopic level. MEP 7 was localized in the Golgi apparatus, in small electron-lucent secretory vesicles, and on microvilli of the principal cells from the distal caput epididymidis to the cauda. The luminal contents were also immunoreactive in these regions of the epididymis. Although some gold particles were associated with the sperm surface, there was no selective concentration of these particles. In addition, MEP 7 was localized in large (600 nm) supranuclear endocytic vesicles and in infranuclear lysosomes. MEP 10 immunoreactivity was also seen on the microvilli of the principal cells of the distal caput and corpus and the luminal contents from the distal caput to the cauda epididymidis. There was no association of gold particles with the sperm surface. In contrast to MEP 7, there was no detectable MEP 10 immunoreactivity on the organelles of the principal cells involved in protein secretion or endocytosis. Clear cells also demonstrated immunoreactivity to MEP 7 and MEP 10. However, the intensity of immunolabeling, and the number of clear cells labeled, was greater with MEP 10 than MEP 7. In the case of MEP 7, the gold particles were located on the large supranuclear endocytic vesicles and on some infranuclear lysosomes, from the proximal corpus to the middle cauda, while in the case of MEP 10, gold particles were predominantly present in infranuclear lysosomes from the distal caput to the middle cauda. These results suggest that the principal cells are involved in both the secretion and endocytosis of MEP 7. The MEP 10 and MEP 7 proteins present in the lumen of the mouse epididymis are endocytosed from the lumen and degraded in the clear cells. However, the process of endocytosis by the clear cells of these two proteins appears to be different.

Secretory vesicle and cell surface markers for human endocrine pancreatic and pituitary tumors.

9 1992 Blackwell Scientific Publications, Inc. The pancreatic islet and the adenohypophysis are endocrine tissues sharing several properties. These organs are both composed of different endocrine cells secreting a variety of hormones. These are stored within the cells in dense core secretory vesicles and are released by appropriate stimuli. In addition to dense core secretory vesicles, endocrine pancreatic and adenohypophyseal cells contain small translucent vesicles, which are very similar to synaptic vesicles not only in their morphological appearance but also with regard to their protein constituents [31, 64]. Indeed, several lines of evidence suggest that pancreatic and adenohypophyseal endocrine cells have an additional secretory pathway for neurotransmitters besides the classic exocytotic pathway for hormone release [31, 54, 60]. Proteins present in the dense core vesicles containing hormones, such as chromogranins and secretogranins, have also been detected in their counterparts within neurons (for refs. see [30, 37, 38, 61]). This indicates that neurons and endocrine ceils are very similar with respect to the composition of their characteristic small secretory vesicles (SSV) and large secretory vesicles (LSV) besides having a variety of molecular, biochemical, and functional similarities [25]. Neurons and endocrine cells also share proteins exposed on the external surface. The best documented proteins of this class are members of the family of neural cell adhesion molecules (NCAMs), which are present in endocrine cells of the adult, including pancreatic islet and adenohypophyseal cells [2, 39-41]. While tumors derived from the endocrine pancreas are rare [35], pituitary tumors are frequently observed [27, 35, 73]. Besides symptoms produced locally by the tumors, overproduction of their hormones and their subsequent effects in the body often permit a straightforward diagnosis. However, a significant percentage of endocrine pancreatic and pituitary tumors do not cause elevated serum hormone levels and thus are hormonally inactive [27, 29, 73]. In such cases additional characteristic constituents of endocrine cells are critical in providing information on the nature, location, and distribution of endocrine tumor cells in the body. Here we summarize recent findings concerning the expression of membrane proteins of SSV and of the cell surface antigen NCAM by normal and neoplastic endocrine cells.

Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells.

Regulated secretory cells have two pathways that transport secreted proteins from the Golgi complex to the cell surface. To identify carrier vesicles involved in regulated and constitutive secretion, PC12 pheochromocytoma cells were labeled with [35S]sulfate to identify markers for the two secretory pathways, then mechanically permeabilized and incubated in vitro. Small constitutive secretory vesicles, containing mostly sulfated proteoglycans, accumulated during an in vitro incubation with ATP. In the presence of GTP gamma S, the constitutive vesicles became significantly more dense, suggesting that a coated intermediate was stabilized. Larger immature regulated secretory granules, enriched in sulfated secretogranin II, also escaped from the permeabilized cells in vitro. During granule maturation, their density increased and the amount of cofractionating proteoglycans diminished. The data suggest that sorting continues during secretory granule maturation.

Growth and characterization of isolated bovine tracheal gland cells in culture. Influence of a reconstituted basement membrane matrix

We describe a method for establishing the culture of bovine tracheal submucosal gland (BTG) cells, in which we have also examined the influence of a reconstituted basement membrane matrix derived from the Engelbreth-Holm-Swarm tumor (EHS) on the growth and morphological differentiation of these cells. BTG cells have been isolated by tissue enzymatic digestion using trypsin, deoxyribonuclease I, elastase, hyaluronidase and EGTA for 1 hr at 37 degrees C. Afterwards, cells and tissue were collected by centrifugation and were incubated for 15 min with 15% newborn calf serum to inactivate the proteolytic enzymes. Enzymatic digestion using only trypsin, centrifugation and inactivation steps were repeated three times. Using this protocol, we obtained 15 +/- 4 (X 10(6] cells per g of tracheal submucosa with 72 +/- 2% (n = 5) cell viability. On microscopic observation, isolated cells were mainly composed of serous type glandular cells. Cells were cultured in a 1:1 medium of Dulbecco's Modified Eagle's/Ham's F12 supplemented with 10% fetal calf serum and subcultured in either plastic flasks or flasks coated with EHS matrix. On the plastic, the BTG cells exhibited at confluency an epithelioid appearance. They stained positively with the immunofluorescent anticytokeratin antibody and contained PAS-staining granules. By electron microscopy, lactoferrin, a protein marker specific to the serous cells, was demonstrated immunocytochemically in small secretory vesicles. BTG cells cultured on EHS matrix revealed a significantly increased growth in comparison to those cultured on plastic. In post-confluent culture of BTG cells on EHS matrix, we observed numerous dome-like structures formed by differentiated cells which were joined together around luminal spaces.

The male copulatory apparatus in an opisthobranch mollusc, Runcina

The copulatory apparatus of a primitive opisthobranch, Runcina, is described. The apparatus is comprised of the following organs: the spermatic bulb, the prostrate, the penis and the penial sac. The spermatic bulb wall consists of cuboidal epithelium with forked microvilli and densely arranged cilia. Prior to copulation the interior is tightly packed with sperm. The prostate is lined with alternating glandular and supporting cells, the latter being compressed but with a mushroom-shaped apex bearing a few forked microvilli and many cilia. The glandular cells produce differing secretions, each cell producing a single type. Large paracrystalline structures enclosed in cells close to the penial area are particularly striking. Considerable amounts of the secretory products are accumulated in the protruding cell apices. One type of inclusion is found at a later stage, packed around the sperm mass within the spermatophore; its function, and the fate of the other secretions is not yet clear. The epithelium of the penis is of more columnar structure, covered with forked microvilli and extremely long cilia which are anchored by long rootlets in the cells. Some of the cells contain large electron-dense secretory granules, others hold accumulations of small secretory vesicles in their apices. It seems likely that these contribute towards the outer layer of spermatophore. The wall of the penial sac is lined by one to two rows of flat-cuboidal cells bearing sparse forked microvilli and cilia.

Cytodifferentiation in the accessory glands of Tenebrio molitor. X. Ultrastructure of the tubular gland in the male pupa.

The tubular accessory gland consists of a simple secretory epithelium surrounded by a muscular coat. Over the pupal instar, the gland increases ten-fold in volume and 15-fold in length. Pupal development is divisible into a phase of mitosis and one of cell growth. During the mitotic phase, cytoplasmic membranes are sparse and nuclei move toward the luminal face of the epithelium to undergo division. In the cell growth phase, the cells become more columnar, a few stacks of rough endoplasmic reticulum are formed, and small dense secretory vesicles appear near the apical surface. The hormonal control of the developmental sequence is discussed.

The secretion of the eggshell of Schistocerca gregaria, analysis of the kinetics of secretion in vitro by light and electron microscope autoradiography.

The secretion of the 2 main layers (endochorion and exochorion) of the eggshell of the desert locust Schistocerca gregaria was investigated using light and electron microscope autoradiography. Follicles undergoing endochorion secretion were labelled using a 3 min 'pulse' of [3H]leucine in vitro followed by a 0-115 min non-radioactive 'chase'. Immediately after the pulse the silver grains were distributed over the cytoplasm and organelles including rough endoplasmic reticulum, while by 2 and 5 min Golgi bodies contained radioactivity. By 12 min from the beginning of the chase the cell apex containing small secretory vesicles was labelled. By 20 min most of the silver grains were over the endochorion. The half-transport time (t50) was 14--15 min (from mid pulse), the lag time was 9--10 min and the percentage transport rate was 14--15% per min. When a 3 min pulse of [3H]galactose was used to label exochorion precursors, the shorter t50 (11 min) and the clumped grain distribution in light microscope autoradiographs after 0-min chase suggested that galactose was incorporated in Golgi bodies. The secretion of exochorion precursors appears to occur at a similar rate to that of endochorion precursors (approximately 15% per min). The results indicate that the follicle cells are among the fastest secreting cells.

Distribution and orientation of microtubules in milk secreting epithelial cells of rat mammary gland.

Quantitative ultrastructural analysis of mid-lactation rat mammary gland demonstrated that cytoplasmic microtubules were present in nearly all secretory epithelial cells examined. Most microtubules were oriented perpendicular to the apical membrane and were found in the apical and medial portions of the cell cytoplasm. There was no statistical difference between the number of microtubules associated with vesicles and the number that were not. Most vesicles which were in contact with microtubules were small (50 to 150 nm), appeared electron lucent and located in a supra-Golgi complex position. Many of these vesicles were seen to be aligned along the axis of longitudinally sectioned microtubules oriented perpendicular to the apical plasma membrane. As measured by a colchicine binding assay, the total tubulin content of mammary tissue from mid-lactation rats was about 107 micrograms/100 mg wet weight. Approximately 19% of the total tubulin was in polymerized form. This study provides evidence that microtubules may be involved in guiding transport of small secretory vesicles to the apical regions of cells for exocytosis.

Ultrastructural and Histochemical Observations on Serous Ovarian Cystadenomas

One hundred and twelve serous ovarian cystadenomas (46 benign, 33 tumours of borderline malignancy and 33 malignant tumours) were investigated with histological and mucin histochemical methods. Electron microscopy was applied to 9 benign, 6 borderline and 5 malignant tumours. The epithelium of the benign serous cystadenomas contained ciliated and nonciliated columnar cells, which sometimes were edematous ("pale cells"), and smaller basal cells. The large Golgi apparatus with small secretory vesicles and occasional larger secretory granules was located in the apical cytoplasm. There were numerous pinocytotic vesicles at the basal plasma membrane. The extracellular mucin in the cystic lumen and the mucin on the apical epithelial border consisted of material with vic-glycols (1.2-hydroxyl groups) mixed with sulpho- and carboxymucin. With increasing degree of malignancy the number of ciliated cells diminished and were not found in the malignant epithelium. The number of nucleoli and the amount of carboxymucin with sialic acid residues increased with increasing degree of malignancy. However, there were no single consistent ultrastructural feature which could be considered as a reliable criterion for malignancy of these tumours in the absence of invasive growth. The findings support the view that serous ovarian cystadenomas are of coelomic origin.

Die Morphologie der Nektarausscheidung bei Bromeliaceen: II. Experimentelle und quantitative Untersuchungen bei Billbergia nutans

Summary Nectar secretion in Billbergia nutans is inhibited by cooling, KCN, dinitrophenol, cytochalasin B, colchicine, deoxyglucose, and different dyes. In active nectaries the dictyosomes of the glandular epithelium are hypersecretory, in inhibited ones there are no or only a few small secretory Golgi vesicles. 90 min after feeding with galactose, melibiose, maltose, raffinose, ribose and deoxyglucose, these sugars appear in the nectar. However, feeding with glucose, fructose or saccharose does not change the relation of these sugars in the secreted fluid. Some of the dyes move up in the secretory cells, obviously through the cell walls, but are not secreted. Amino acids are also retained. Provided that all nectar passes the Golgi apparatus, the life-time of a Golgi vesicle is 2,3 min. If all prenectar passes the plasmodesmata between the subepithelial and the epithelial cells, there is a sugar flux of about 2,4 · 10− 18 M saccharose × sec− 1 per plasmodesmos. It seems, however, probable that nectar components also move through the apoplast.

Ultrastructure, composition, and biogenesis of the encapsulation material surrounding the endophyte in Alnus crispa var. mollis root nodules

The encapsulation material surrounding the endophyte of Alnus crispa var. mollis Fern. root nodule was studied by means of a variety of staining methods in light and in electron microscopy. The enzymatic degradation of the capsular material was obtained and the degradation products were studied by thin-layer chromatography. The endophyte capsule was composed mainly of granular and fibrillar non-sulfated and de-esterified polygalacturonic acid or its salts. An endophyte pectic capsule was also demonstrated in 11 additional species of non-leguminous root nodules. Small secretory vesicles, containing electron-dense material similar to that of the endophyte capsule, occurred in free multivesicular bodies throughout the host cell cytoplasm. Ultrastructural observations are consistent with the view that these bodies migrate to the host membrane envelope surrounding the endophyte, fuse with it, and deposit the contents of the small vesicles toward the endophyte cell wall. These small secretory vesicles appeared to originate as blebs from cisternae tips; dictyosomes were abundant in the infected cortical cells. The possible significance of the pectic capsule in the host–endophyte nutritional interactions is discussed.

Effect of calcitonin on rabbit thyroid glands.

Long-term im administration of porcine calcitonin to rabbits in large doses (100 MRC U/kg body weight as total dose) induces a striking proliferation of thyroid cells. Clusters of interfollicular cells disrupting the follicular pattern and predominance of small follicles lined by high columnar cells with infoldings are evident. Ultrastructurally, most of the thyroid follicular cells are in an apparent hypersecretory state, showing an increase in number and size of microvilli, colloid droplets and heterogeneous dense granules as well as a hypertrophy of Golgi complex. A hyperplasia and hypertrophy of parafollicular (C) cells are also observed. These cells display a large population of small dense granules, free ribosomes and secretory vesicles. These findings suggest that chronic calcitonin administration exerts a goitrogenic effect on rabbit thyroids and also inhibits the release of calcitonin from the parafollicular cells. (Endocrinology 90: 1046, 1972)