Distribution of sialoglycoconjugates on the luminal surface of the endothelial cell in the fenestrated capillaries of the pancreas

Sialic acid-bearing molecules on the luminal surface of the vascular endothelium in mouse and rat pancreatic capillaries were detected electron microscopically by using a procedure with ferritin hydrazide (FH), after preferential oxidation of sialyl residues with sodium periodate. The distribution of FH on the endothelial surface demonstrated the existence of microdomains with various densities of sialoglycoconjugates oxidizable by sodium periodate and accessible to the tracer. On the plasmalemma proper, FH binding sites were heterogeneously distributed. Their concentration on various microdomains decreased as follows: plasmalemma proper greater than coated pits greater than stomal diaphragms of plasmalemmal vesicles and transendothelial channels, and fenestral diaphragms. The membrane of plasmalemmal vesicles and transendothelial channels was not labeled by FH. Nonspecific binding of FH to the nonoxidized endothelial surface or that oxidized after neuraminidase treatment was relatively low.     

Some morphological and histochemical aspects of nemertean connective tissue

Summary The organization of the connective tissue system in three nemertine species, Amphiporus pulcher, Lineus bilineatus and Lineus ruber has been studied. Most attention has been paid to A. pulcher. Light microscopical, histochemical and electron microscopical methods have been employed.Three elements have been found and described: several types of free cells, filaments and ground substance. The filaments belong to the collagen family and form a supportive collagen skeleton in the animals. The ground substance is abundant in A. pulcher and is composed of protein and acid and neutral polysaccharides. The connective tissue cells have been classified in three groups, but intergrading between the cells, especially groups 1 and 3 is found. The connective tissue cells are highly polymorphic and often combine several functions in the same cell, e.g. synthesis of extracellular products, phagocytosis, pigment synthesis or uptake and a function as cellular material for regenerative processes.It is stressed that the connective tissue system probably forms a unity in the animals and no attempt has been made to make a rigid and presumably rather unnatural classification into various types.Comparative aspects of nemertine connective tissue have been discussed in relation to the patterns found in acoel, triclad and polyclad turbellarians. It is concluded that the nemertean connective tissue system still has features in common with turbellarian patterns, especially the one found in polyclads. However, the nemertine connective tissue system exhibits greater complexity than those found in turbellarians. The nemertine connective tissue system both shows continuity to the turbellarian organizations but also has features added so that it conforms with the patterns found in most groups of animals including vertebrates.The author is indebted to Professor G. Thorson and Dr. Gunnar Berg, The Marine Biological Laboratory, Elsinore, for supplying the animals. The efficient and conscientious assistance of Mrs. K. Bahnert and Miss Åse Madsen is greatly appreciated.     

Some observations on cell wall structure and taxonomy ofPhymatodocis nordstedtiana (Conjugatophyceae,Chlorophyta)

Filaments ofPhymatodocis nordstedtiana Wolle were isolated from a sample of a Texan lake. Cultures were established and examined by light and scanning electron (SEM) as well as transmission electron microscopy (TEM). It is shown that the pores apparent on light microscopical examination are not of the cosmaroid type as expected. TEM examination disclosed that they are similar to those found in the generaClosterium Ralfs andPenium Bréb. Furthermore, it could be demonstrated by light and SEM microscopy that the primary cell wall is shed during cell division. The remaining secondary cell wall of the mature cell consists of interwoven bands of parallel microfibrils. A conspicuous overlap of the semicell walls clearly denotes the isthmus region. The significance of these deviations unusual for desmids is discussed. Suggestions are made that the taxonomic position ofPh. nordstedtiana should be re-evaluated.     

Morphological study of lectin binding in the rabbit temporomandibular joint disc

Summary Glycoconjugates of the extracellular matrix are important for the normal mechanical functions of connective tissue structures such as the temporomandibular joint disc. Since lectins are known to bind to sugar residues with high affinity, a variety of lectins were used to study the presence and distribution of glycoconjugates in the temporomandibular joint disc. Discs were removed from 6 to 8-month-old rabbits and either sectioned in a cryostat and processed for light microscopy or fixed in 2% glutaraldehyde and processed for electron microscopy. The frozen sections were incubated with fluorescein- or peroxidaseconjugated lectin solutions. Ultrathin sections mounted on grids were incubated with lectins combined with a colloidal gold marker system for electron microscopical study. Our results indicate thatCanavalia ensiformis agglutinin (ConA) showed little or no binding to the discal tissue.Triticum vulgaris agglutinin (WGA) andMacluras pomifera (MPA) were bound strongly to both the synovium and the extracellular matrix and WGA also bound to the territorial matrix of chondrocyte-like cells.Glycine max andArachis hypogoea agglutinins (SBA and PNA), were localized in the synovium and extracellular matrix but to a lesser degree than WGA and MPA. WGA, MPA,Griffonia simplicifolia II andUlex europaeus were bound by discal fibroblasts. WGA was also localized in lysosomes of synovial A-cells (macrophages). The electron microscopical studies with lectins and colloidal gold marker systems indicated that some areas of the disc may be fibrocartilagenous as had been suggested by earlier immunohistochemical studies using monoclonal antibodies to characteristic glycosaminoglycans (GAGs) in cartilage.     

Reassembly of the Regularly Arranged Subunits in the Cell Wall of Lactobacillus brevis and Their Reattachment to Cell Walls

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in the native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L. plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.     

Elektronenmikroskopische Analyse der Musterbildung im Antheridium derPolypodiaceae

An electron microscopical investigation of the cell walls in young antheridia ofPolypodium crassifolium andPlatycerium alcicorne confirms the classical developmental model as postulated byStrasburger and byKny. The structure of the basal cell walls, both of the funnel cell and of the operculum, and especially the evidence of plasmodesmata in those walls, disprove the widely accepted interpretation presented 1951 byDavie.

     

Lectin-binding patterns on the plasma membranes of dissociated rat liver cells

Summary Carbohydrate moieties on the surface of dissociated rat liver cells were examined electron microscopically using ferritin-or horseradish peroxidase (HRP)-conjugated lectins as probes. Rat liver was fixed by perfusion with 0.7% glutaraldehyde via the portal vein and dissociated into single cells with gentle homogenization. Concanavalin A (ConA), Ricinus communis agglutinin (RCA), and wheat germ agglutinin (WGA) bound almost evenly to the entire cell surface of hepatocytes as well as of endothelial cells. Ulex europaeus agglutinin I (UEA-I) and peanut agglutinin (PNA) revealed no binding to any region. Dolichos biflorus agglutinin (DBA) was found to bind exclusively to the sinusoidal surface of hepatocytes and to endothelial cell surfaces. Soybean agglutinin (SBA)-binding was restricted to the endothelial cell surfaces and part of the sinusoidal microvilli of hepatocytes. Regional differences in lectin-binding pattern were visualized between the sinusoidal and the lateral or bile-canalicular surfaces of the hepatocytes. A polarity may exist on the hepatocyte cell surfaces in terms of the distribution pattern of the carbohydrate moieties, especiàlly those of N-acetylgalactosamine.     

Real-time monitoring of extracellular matrix-mediated PC12 cell attachment and proliferation using an electronic biosensing device

The attachment and proliferation of a well-established, neuron-like cell line, rat pheochromocytoma (PC12) cells, on different extracellular matrices (ECMs) was monitored using cellular impedance sensing (CIS). Commonly used ECMs, including fibronectin, laminin, poly-l-lysine, collagen and poly-l-lysine followed by laminin, in addition to DMEM cell culture media alone as a control, were studied: CIS identified the dynamic progress of the adhesion and proliferation of the cells on different ECMs. Among these modified ECM surfaces, the laminin- and poly-l-lysine/laminin-modified surfaces were the best suited for the neuron-to-electrode surface attachment and proliferation, which was confirmed by MTT assays and a scanning electron microscopy analysis. This work provides a simple method to study neuron cell/ECM interactions in a real-time, label-free, and quantitative manner.     

Inhibition of endothelial cell morphogenetic interactions in vitro by alpha- and beta-xylosides

Summary Bovine aortic endothelial cells retain the ability to undergo histotypic morphogenetic interactions in vitro as evidenced by a) the reversible expression of a sprouting cell phenotype and b) the patterned self-association of these sprouting cells into three-dimensional meshworks and tubule-like structures. These morphogenetic events are inhibited by xylosides in a dose-dependent manner. Two types of beta-xylosides (p-nitrophenyl-beta-d-xylopyranoside and 4-methylumbelliferyl-beta-d-xylopyranoside) and one alpha-xyloside (p-nitrophenyl-alpha-d-xylopyranoside) were tested. Beta-xylosides are well characterized acceptors of glycosaminoglycan chains, whereas alpha-xylosides do not function in this capacity and have been extensively used as negative controls when studying the effects of beta-xylosides. Both alpha-and beta-xylosides inhibited endothelial morphogenetic interactions. This inhibition was slowly reversed during the 6- to 7-d period following removal of the xyloside. Inhibition of morphogenetic interactions by xylosides occurred at concentrations (0.5 to 2.0 mM) that had no demonstrable effects on cell proliferation, migration, or adhesion to 2-D plastic or collagen substrata. The xylosides seemed to inhibit cell spreading on a 3-D environment, they also inhibited the incorporation of [³H]-proline and Na₂ ³⁵SO₄ into the extracellular matrix deposited by the cells, suggesting that the inhibition of morphogenesis may be related to the inhibition of matrix deposition. Endothelial morphogenetic interactions were not inhibited by the extracellular matrix or by the conditioned medium produced by cells cultured in the presence of xylosides.     

Scanning electron microscopic studies of cilia

Summary A simple method for the preparation of ciliated epithelia for study with the scanning electron microscope is described. Ciliary groups are well preserved and it is possible to discern individual cilia and work out their numbers and orientation. Following scanning electron microscopical study some of the material was prepared for transmission electron microscopy and the ultrastructure of the tissue was found to be surprisingly well preserved. The tracheal epithelium of the rabbit, the olfactory epithelia of the goldfish and the rabbit, and the sensory epithelia in the statocyst of a cephalopod mollusc were examined with the scanning electron microscope to demonstrate the possibilities of the method. Acknowledgements. We would like to thank Professor J. Z. Young for his continued interest and support. The scanning electron microscope was purchased with a grant provided by the Science Research Council to Dr. Boyde, Mr. R. Willis helped in the initial stages of the study, Mr. G. Savage provided help with the goldfish material, Mr. S. Waterman provided much photographic assistance, and Mrs. N. Finney the secretarial assistance.     

Studies on growth inhibition by lectins of penicillia and aspergilli

It has previously been shown in our laboratory that wheat germ agglutinin (WGA) binds to Trichoderma viride and inhibits growth of this fungus. Here we report on the effect of WGA, soybean agglutinin (SBA) and peanut agglutinin (PNA) on Penicillia and Aspergilli. Binding of the lectins to the fungi was examined with the aid of their fluorescein isothiocyanate (FITC) conjugated derivatives. FITC-WGA bound to young hyphal walls of all species, in particular to the hyphal tips and septa, in agreement with the chitinous composition of the cell walls of the two genera. Hyphae of all species examined were labelled, though in different patterns, by FITC-SBA and FITC-PNA, suggesting the presence of galactose residues on their surfaces. Young conidiophores, metulae (of the Penicillia), vesicles (of the Aspergilli), sterigmata and young spores, were also labelled. The three lectins inhibited incorporation of [³H]acetate, N-acetyl-D-[³H]glucosamine and D-[¹⁴C]galactose into young hyphae of Aspergillus ochraceus, indicating interference with fungal growth. Inhibition of spore germination by the three lectins was also observed. Preincubation of the lectins with their specific saccharide inhibitors prevented binding and the inhibitory effects. We conclude that lectins are useful tools for the study of fungal cell surfaces, and may also serve as an important aid in fungal classification. The present findings also support the suggestion that one role of lectins in plants is protection against fungal pathogens.Abbreviations Con A concanavalin A - PNA peanut agglutinin - SBA soybean agglutinin - WGA wheat germ agglutinin - FITC fluorescein isothiocyanate - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine     

An osmiophilic bilaminar lining film at the respiratory surfaces of avian lungs

Summary The ultrastructure of the lungs of an adult pigeon, sparrow and hen, and of foetal chickens and chicks of different ages were studied. The presence of a noncellular continuous film on the air capillary surfaces, i.e. on the effective respiratory surfaces, was established at all stages of development as well as in three observed avian species. This film was bilaminar, of a thickness of approximately 100–150 Å. The upper and lower layers are strongly osmiophilic, the intermediate layer is of low electron density. This membrane is lacking in all respiratory passages with the exception, mentioned above, of air capillaries. The lung tissue was examined by the periodic acid-silver methenamine technique for the demonstration of polysaccharides and by the colloidal iron method (Mowry's modification of Hale's method) for the detection of acid mucopolysaccharides. After the first mentioned reaction, the picture corresponding to the visualization of carbohydrate-rich cell coat (Rambourg, 1967) was obtained. The reaction with colloidal iron was strongly positive at the air capillary surfaces. Nevertheless, the weakly expressed positive reaction was also present at the endothelial cell surfaces. It was assumed that the bilaminar osmiophilic lining film did not contain polysaccharides. Furthermore, this film was also shown to be present in the chicken's lung which had been rinsed several times with saline before fixation. It seems that this layer is secreted by the air capillary epithelial cells. We consider the type of secretion to be merocrine. In view of its behaviour in response to certain histochemical techniques and on the basis of electron microscope investigation its lipoproteinous nature is most probable.     

A neurofibrillar body in the perikaryon of nervus terminalis ganglion cells in teleosts

Summary In three species of Teleosts (Tinea tinea L., Leuciscus cephalus cabeda Risso, Epinephelus guaza L.) a round strongly argentophilic body of considerable size occurs in the cytoplasm of the nervus terminalis ganglion cells. In Tinea, surgical interruption of functional connections of the ganglion cells does not produce any apparent change either in the number or in the size of these cytoplasmic bodies.Electron microscopical observations show that the neurofibrillar body is made up of densely packed and irregularly arranged bundles. In cross section, each of these bundles appears to be composed of neurofilaments (100 Å in thickness) and neurotubules (diameter: 300 Å). Each tubule is surrounded by 9–10 filaments equi-distant from one another, and at a distance of 30–40 Å from the central tubule.The authors are indebted to Prof. G. Palladini for helpful histochemical advice, to Prof. B. Bertolini for electron micrographs and to Mr. D. Scorsini for skilful technical assistance.     

Lectin histochemical studies of mouse granulated metrial gland cells

A lectin histochemical study has been carried out on mouse granulated metrial gland cells, the major leucocyte population that differentiates in the uterine wall in pregnancy. The binding characteristics of 26 lectins were examined using light microscopical methods. Fourteen of the lectins, with affinities ranging through N-acetylgalactosamine, galactose, N-acetylglucosamine, mannose and sialic acid residues, bound to the cytoplasmic granules of granulated metrial gland cells, and each appeared to bind to the limiting membrane of the granules. The binding characteristics of three of these lectins (Wheat germ agglutinin, Concanavalin A and Helix pomatia agglutinin) were examined using electron microscopical methods. These showed a different binding pattern to the cytoplasmic granules of granulated metrial gland cells compared with that found using light microscopical methods, as they appeared to bind evenly across the granule's matrix. This binding pattern corresponds to the reactivity of the granule matrix in the periodic acid--Schiff technique. Six lectins bound to the cell membranes of granulated metrial gland cells. These included the E and L isoforms of Phaseolus vulgaris agglutinin, with affinities for complex carbohydrates, whose binding differences were related to the stage of differentiation of the granulated metrial gland cells. The lectin binding described presents additional markers of granulated metrial gland cells and tools for investigating carbohydrate moieties in the functional activities of granulated metrial gland cells This revised version was published online in November 2006 with corrections to the Cover Date.     

Binding of concanavalin A and wheat germ agglutinin by murine peritoneal macrophages: ultrastructural and cytophotometric studies

Summary Concanavalin A and wheat germ agglutinin were employed in conjunction with the horseradish peroxidase-diaminobenzidine method for the detection of sugar residues on the surface coat of exudate and resident murine peritoneal macrophages. Electron microscopical and cytophotometric techniques were used for the visualization and quantification of the final reaction product on the surface of cells. After incubation with concanavalin A and wheat germ agglutinin, both exudate and resident macrophages showed readily detectable final reaction product indicating the presence of numerous, easily accessible, -methyl-d-mannosyl andN-acetyl-d-glucosaminyl residues on their surface. The binding of concanavalin A was higher with resident than with exudate macrophages. With wheat germ agglutinin, a different pattern of lectin binding was observed: more electron-dense product was deposited on exudate than on resident macrophage surfaces. The binding of concanavalin A and wheat germ agglutinin to macrophages was inhibited by the competing sugars -methyl-d-mannoside andN-acetyl-d-glucosamine, respectively.     

Cloning and Structural Analysis of a Haemocyanin from the Stonefly Perla grandis

We characterized two subunits of a putative haemocyanin from the stonefly species Perla grandis. In particular, we cloned and sequenced the corresponding cDNAs and studied their expression in different insect stages. Moreover, using the deduced amino acid sequences, homology studies were performed both on their primary and tertiary structures. 3-D molecular modelling data showed that the residues involved in the oxygen transport and subunits contacts were located in spatial positions preserving the functionality of the molecule. Despite it was paradigmatically affirmed that insects do not have respiratory proteins, our data suggest that the haemocyanin could be involved in the respiratory mechanisms of P. grandis. As far as we know, this is the first haemocyanin 3-D structure described and analyzed in insects.     

Haemocyanin oxygen binding and the physiological ecology of a range of talitroidean amphipods (Crustacea) I. pH,temperature, and l-lactate sensitivity

Summary The effect of pH, temperature, and l-lactate on the O₂ bindign properties of haemocyanin (Hc) from three talitroidean species i.e., the aquatic Apohyale pugettensis, the semi-terrestrial Megalorchestia californiana, and the semi-/euterrestrial Traskorchestia traskiana was studied. The Hc of A. pugettensis was characterized by a higher O₂ affinity (and more pronounced Bohr shift) than the Hc of either M. californiana or T. traskiana. Apohyale was the only species that possessed He that was sensitive to temperature change. Resuspending Hc from each of the three species in a stock Ringer's solution (based on the ionic composition of the haemolymph of T. traskiana) showed that the persistence of the difference in Bohr shift between Apohyale and the other two species was due to differences in the haemocyanins themselves and not attributable to their respective ionic environments. An inverse relationship was found between the cooperativity (n ₅₀) and pH of Hc from T. traskiana and A. pugettensis but not for M. californiana. In each case adding l-lactate increased Hc O₂ affinity, but this was most pronounced for A. pugettensis.Abbreviations Hc haemocyanin - STR stock Traskorchestia Ringer's solution     

Post-embedding localization of glycoconjugates by means of lectins on thin sections of tissues embedded in LR white

Summary A simple post-embedding technique for the electron microscopical detection of lectin-binding sites using thin sections of tissues embedded in the resin LR White is described. With this technique, no prior etching of the sections is necessary. The cellular fine structure is well preserved and permits close correlation of the labelling to distinct cellular compartments. After mild aldehyde fixation (4% formaldehyde and 0.5% glutaraldehyde for 30 min), enterocyte brush border, vesicles and lysosomes as well as goblet cell Golgi apparatus and mucin are intensely stained after 30–60 min.The hydrophilia and penetrability of LR White is shown by the formation of oxidized diaminobenzidine reaction product arising from horseradish peroxidase-conjugated lectins. The precipitate not only covers the surface of the sections but is also formed within the resin, as is revealed on cross-sections through thin and semithin sections. The addition of 0.2m solutions of the appropriate inhibitory sugars prevented staining, which indicates a specific binding. Examples are given of the binding of gold-, ferritin- and peroxidase-conjugated lectins for the purpose of detecting glycoconjugates in various intracellular compartments.     

Development of a freeze substitution technique to examine the structure ofLactobacillus casei GR-1 grown in agar and under batch and chemostat culture conditions

A morphological examination was undertaken ofLactobacillus casei GR-1 by a freeze substitution technique developed to prevent condensation upon fixation and to preserve extracellular material surrounding the cell wall. The strain was cultured for 24 h in 5% CO₂ at 37°C initially in brain heart infusion agar supplemented with 2% yeast extract, and the cells formed a short, electron-dense, tightly bound capsule observed under electron microscopy. The cell wall structure was resolved in most cases. Batch cultures were then established by use of pooled human urine with and without addition of lactose and glucose. Examination of the bacteria demonstrated less compact, but more fibrous extracellular material surrounding the cells in a less uniform fashion. The lactobacilli were then grown under nitrogen-and carbonlimited conditions in a chemostat continuous culture system. The nitrogen-limited cells formed a tightly bound, uniform, and electron-dense capsule, while the capsule of the carbon-limited cells appeared longer, more fibrous, but less electron dense in nature. The results indicate that nutrient conditions affect the morphology of lactobacillus and verify that the freeze substitution technique is a useful method to analyze the structure of bacterial cell surfaces. The importance of nutritional changes in the microbial ecology of the urogenital tract can be uncovered by examining these organisms with different culture techniques combined with freeze substitution and electron microscopy.     

Molecular characterization and evolution of haemocyanin from the two freshwater shrimps Caridina multidentata (Stimpson, 1860) and Atyopsis moluccensis (De Haan, 1849)

Haemocyanin (Hc) is a copper-containing respiratory protein, floating freely dissolved in the hemolymph of many arthropod species. A typical haemocyanin is a hexamer or oligohexamer of six identical or similar subunits, with a molecular mass around 75 kDa each. In the crustaceans, the haemocyanins appear to be restricted to the remipedes and the malacostracans. We have investigated the haemocyanins of two freshwater shrimps, the Amano shrimp Caridina multidentata and the bamboo shrimp Atyopsis moluccensis. We obtained three full-length and one partial cDNA sequences of haemocyanin subunits from the Amano shrimp, which were assigned to the α- and γ-types of decapod haemocyanin subunits. Three complete and two partial haemocyanin cDNA sequences were obtained from the bamboo shrimp, which represent subunit types α, β and γ. This is the first time that sequences of all three subunit types of the decapod haemocyanins were obtained from a single species. However, mass spectrometry analyses identified only α- and γ-type subunits, suggesting that a β-subunit is not a major component of the native haemocyanin of the bamboo shrimp. Phylogenetic and molecular clock analyses showed that malacostracan haemocyanins commenced to diversify into distinct subunit types already ~515 million years ago. β-subunits diverged first, followed by α- and γ-type subunits ~396 million years ago. The haemocyanins of phyllocarids and peracarids form distinct clades within the α/γ-cluster. Within the Caridea, an early divergence of distinct α-type subunits occurred ~200 MYA. The tree of the γ-subunits suggests a common clade of the Caridea (shrimps) and Penaeidae (prawns).