Ultrastructural localization of Helix pomatia lectin-binding sites in mouse lung elastic fibers

Summary Helix pomatia (Snail) lectin complexed with colloidal gold (HPL-gold) recognized binding sites on elastic fibers in plastic embedded sections of lung tissue from mice of several ages. Deposition of the lectin-gold particles was examined by electron microscopy. Structures such as the elastic laminae of pulmonary vessels and elastic fibers throughout the lung was specifically and intensely decorated by the HPL-gold complex and easily visualized. The binding of the HPL-gold particles was primarily to sites on the amorphous component of elastin, to the virtual exclusion of the microfibrillar elastin elements, collagen fibers and other components of the extracellular matrix. In addition, moderate age differences in the binding of HPL-gold to elastin were apparent. These observations appear to be the first demonstration of the presence, in the amorphous component of elastin, of glycoconjugates that are specifically recognized by HPL and suggest a method by which the involvement of glycoconjugates in lung elastogenesis could be explored.Supported in part by USPHS Grant HL-32870     

Ultrastructural localization of lectin-binding sites in different basement membranes

Summary In the present work we localized binding sites for the lectins WGA, RCA I, con A and SBA at the ultrastructural levels in morphologically different basement membranes. These different basement membranes included (a) thin ones, for example, tubular basement membrane of the mouse kidney which separates epithelial cell layers from mesenchymal cells and glomerular basement membrane which separates epithelial cells from other epithelial cells, (b) thick multilayered ones, for example, Reichert's membrane which is built up during the embryonic development of rodents and as an example of a pathologically thickened basement membrane, the basement membrane of the Engelbreth-Holm-Swarm (EHS) sarcoma. We were able to show that, in contrast to the thick multilayered basement membranes, the thin ones showed a strong positive SBA-binding pattern. Thick basement membranes otherwise revealed very strong labelling with the lectins WGA and RCA I. Our findings lead us to conclude that thin and thick basement membranes differ markedly in the quality and quantity of the carbohydrates which they contain.     

Ultrastructural localization of carbonyl reductase in mouse lung

Summary The immunocytochemical localization of tetrameric carbonyl reductase in the mouse lung was determined by an electron-microscopical immunogold procedure using monospecific antibodies against the enzyme. The labelling of carbonyl reductase was observed within the mitochondria of the ciliated and non-ciliated cells of the bronchioles and the type II alveolar pneumocytes, and the density of labelling in the non-ciliated cells was higher than those in the other cells. No significant labelling was detected over other compartments of the epithelial cells. The labelling was undetectable in the type I alveolar cells, alveolar macrophages and connective tissue cells of the lung. These results clearly indicate the localization of carbonyl reductase to the mitochondrial matrix of these epithelial cells, of which the non-ciliated bronchiolar cells contained particularly high amounts of the enzyme.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Anatomic distribution of lectin-binding sites in mouse testis and epididymis

Abstract. Testis and epididymis of sexually mature mice were studied histochemically using 25 fluorescein-isothiocyanate-labeled lectins. Several lectin-specific binding patterns were recognized. Thus, HAA, HPA, GSA-I, and UEA-I1 reacted only with spermatozoa. PNA, GSA-11, SBA, VVA, BPA, RCA-I, and RCA-I1 reacted with spermatozoa and spermatocytes. WGA, PEA, LCA, and MPA reacted with spermatogonia, spermatocytes, and spematozoa in increasing order of intensity. ConA, SUC. ConA, LAA, STA, LTA, LPA, PHA-E, PHA-L, IJEA-I, and LBA reacted with all spermatogenic cells with equal intensity. In the epididymis, 12 lectins reacted uniformly with the epithelial cells lining all segments of this organ. One lectin (VVA) did not react with epididymal lining cells. The remaining 12 lectins reacted in a specific manner with portions of the head, body, or tail, thus selectively outlining different portions of the epididymis. RCA-I and RCA-I1 selectively accentuated the so-called halo cells of the epididymis. These findings provide a detailed map of lectin-binding sites in the mouse testis and epididymis and show that certain lectins can be used as specific markers for spermatogenic cells and segments of the epididymis.     

Ultrastructural distribution of lectin-binding sites on gastric superficial mucus-secreting epithelial cells

Normal human gastric epithelial cells were examined by electron microscopy using each of five biotinylated lectins [Ulex europaeus agglutinin I (UEA-I), peanut agglutinin (PNA), wheat germ agglutinin (WGA), soybean agglutinin (SBA) andDolichos biflorus agglutinin (DBA)] as a probe. We employed 35 gastric surgical specimens removed from complicated peptic disease. The lectin-binding sites were revealed with streptavidin-colloidal gold complex. All specimens were embedded in Spurr and LR White resins. In superficial foveolar epithelial cells, the lectins used were generally positive in all cell types (mainly UEA-1 and PNA) on the Golgi region and mucus cytoplasmic vacuoles, with many variations among cells in the same case. On the other hand, extracellular mucus was negative for WGA. Labelling with PNA revealed a biphasic pattern (peripheral positivity) on mucous droplets in surface and foveolar cells. Thecis side of the Golgi apparatus was labelled with SBA and PNA and rough endoplasmic reticulum with SBA (only five cases). Lectin-binding variability could be related to heterogeneous composition of gastric mucus. Our results with SBA suggest initiation ofO-glycosylation at the Golgi apparatus; however a role of the rough endoplasmic reticulum cannot be excluded (N-glycosylation). We propose the following sequence of sugar addition to the carbohydrate side-chains of gastric glycoproteins: (1) GaNAc (Golgi apparatuscis-side), (2) GlcNAc (Golgi apparatus intermediate face), (3) GalNac or Gal, -l-fucose (Golgi apparatustrans-side).Supported by a grant from Junta de Andalucía (Consolidación de Grupos de Investigación. Ref. 541A.6.60.609.018311)     

Ultrastructural study of the shell-repair membrane in the snail,Helix pomatia L.

Summary The shell-repair membrane of the snail, Helix pomatia, has been studied with the transmission electron microscope (TEM). The ultrastructure of the repair membrane, in the initial stages of calcification, revealed the presence of a fibrillar protein, proteoglycan granules, osmiophilic vesicles, and cytoplasmic dense bodies of different size and structure. The involvement of the cell constituents in the formation of calcifying centra and initial crystal formation is discussed. The amoebocytes present within the repair membrane appeared to be involved in three different functions: (1) phagocytosis, (2) release of granules, vesicles and dense bodies, and (3) secretion of a fibrillar protein. The possible lytic function of the amoebocytes is mentioned. The common features in the mineralizing process of the shell-repair membrane of the snail and the epiphyseal cartilage of the mammals were noted.This investigation was supported by a grant from the Swedish Natural Science Research Council, which is gratefully acknowledged. I am indebted to Miss Ch. Stensjö and Mrs. E. Hellmén for their technical assistance     

Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, the lungs of mice ranging from the late fetal stage (17 days of gestation) to the puberal stage (27 days) were surveyed for their proteoglycans. A large Cuprolinic Blue-positive filament is present within the connective tissue of lungs of late fetal and young postnatal mice. It is mostly located at the boundary between large extracellular matrix structures and electron microscopically empty areas, but sometimes also at the surface of fibroblast-like cells. The stainability of the filament disappears after treatment with chondroitinase ABC or chondroitinase AC, but not after treatment with nitrous acid. The Cuprolinic Blue-positive structure appears to be most abundant around 2 days postnatally. From day 10 on, its number decreases dramatically, and it can be no longer observed in the lungs of 27-day-old mice.     

Identification of CO2 Chemoreceptors in Helix pomatia

SYNOPSIS. Gas exchange in pulmonate snails of the family Helicidaeoccurs through a highly vascularized diffusion lung known asthe mantle. The extent of ventilation of the mantle dependsupon the duration and size of opening of an occlusible poreknown as the pneumostome. In Helix aspersa and Helix pomatia,pneumostomal size and frequency of opening are exquisitely sensitiveto CO₂. Respiratory CO₂ chemosensitivity resides in a discreteregion of the subesophageal ganglia. The discharge pattern ofmany neurons in the chemoreceptor area changes during stimulationwith CO₂. However, the electrophysiological response to CO,stimulation alone does not discriminate between CO₂ chemoreceptorcells and CO₂-insensitive neurons active in the pneumostomalresponse to CO₂. We identified a subset of CO₂-sensitive neuronsfrom the larger population of neurons active during CO₂ stimulation.The action potential discharge frequency of CO₂ chemosensoryneurons increased in response to CO₂ stimulation. An increaseddischarge frequency of CO₂-sensitive neurons was associatedwith increased pneumostomal opening, and both the size and thefrequency of pneumostomal opening increased during CO₂ stimulation.Injecting depolarizing current into individual chemosensoryneurons elicited opening of the pneumostome in the absence ofCO₂. Action potential generation in response to CO₂ was independentof synaptic transmission. Removal of individual CO₂-sensitivecells or inhibition of action potential generation in CO₂-sensitivecells reduced or eliminated pneumostomal responses to CO₂. CO₂sensitivity in chemoreceptor cells required extracellular calcium,but not sodium. Substituting barium for calcium supported chemoreceptoractivity. In summary, we have identified respiratory related,chemosensory neurons that are CO₂ sensitive in the absence ofsynaptic input.     

Parvalbumin-immunoreactive neurons in the brain of Helix pomatia

Parvalbumin-immunoreactive material was detected in the central nervous system of the snail, Helix pomatia. Each ganglion investigated contained parvalbumin-immunoreactive neurons. The molecular weight of Helix parvalbumin-immunoreactive material as determined by Western blots is about 40 kilodaltons. ⁴⁵Ca²⁺ overlays showed that this protein binds Ca²⁺. In contrast to vertebrates, in Helix neurons parvalbuminlike material was not colocalized with the neurotransmitter gamma-aminobutyric acid (GABA).     

Nerve cells of Helix pomatia during hypercapnia

Hypercapnia leading initially to activation of synaptic activity and of pace-maker mechanism is accompanied later by inactivation of the electro-excitable membrane and is completed by loss of chemosensitivity. Restoration of the properties of the neurone following transition to normal atmospheric air develops in reverse order. All hypercapnic changes of the properties of the electroexcitable and chemosensitive membrane take place at a constant level of the membrane potential which points to stability of K-Na-pump.     

Food-attraction conditioning in the snail,Helix pomatia

Adult pulmonate snails (Helix pomatia) were released equidistant between two types of food, carrot and potato, respectively. Naive snails moved in different directions and did not locate either food above chance, although both foods were readily eaten upon direct contact. After a single carrot feeding episode, 75% of the carrot-fed snails moved directly towards the carrot and ate it. Conversely, potato-fed snails located the potato in 67% of the cases. Snails that were fed apple or lettuce behaved like naive animals, with the majority of animals (75% in both cases) locating neither the carrot nor the potato.The ability of snails to locate this particular food after a single feeding episode was maintained for at least 11 days, provided that the snails were not exposed to other foods in the interim. If the animals were fed a different food (but still tested for food-finding ability to the initially conditioned food) their orientation preference decreased gradually over a period of 5 days.Although the snails' orientation is based upon olfactory cues, exposure to food odor alone is not sufficient to enable food-finding; additional feeding related stimuli are necessary.These findings indicate that Helix do not possess a predisposition for the foods tested, and further suggest that processes underlying food-finding and food selection are strongly influenced by learning experiences. The conditioning phenomenon underlying food-finding behavior has been called Food-Attraction Conditioning, and appears to be a crucial link between the ecologies of learning and foraging behaviors. The accessibility of the snail's nervous system should permit neuronal analysis of the mechanisms underlying such a unique and complex learning phenomenon.     

Ultrastructural localization of lectin-binding sites and laminin-like immunoreactivity in glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts

Summary Lectins with different sugar specificities and labeled with horseradish peroxidase or gold were used to study, at the electron-microscopic level, surface glycoconjugates of glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts. Differential binding to differentiating glial cells and to neurites was demonstrated. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) bound to glial and neurite surfaces with different degrees of labeling. The formation of glial processes and junctional complexes was invariably accompanied by a corresponding increase of Con A- and WGA-receptors. Peanut agglutinin (PNA) failed to bind to glial cells but strongly stained the plasma membrane of neurite junctions. Lotus tetragonolobus a. (LTA) did not bind either to glial cells or to neurites. In addition, staining with an antibody against laminin showed labeling in areas of neurite outgrowth and neurite interactions; this resembled the localization of PNA receptors. These findings provide evidence for the presence of different carbohydrates at the surface of neurites and glial cells of locust. Their predominant localization in glial processes and neurite junctions suggests that these carbohydrates constitute part of a group adhesion glycoproteins that also includes laminin.