Distribution of lectins in membranes of soybean and peanut plants

The distribution of lectin activity in soybean and peanut plants has been investigated. In both plants activity is found in all tissues examined (roots, shoots and leaves) at all stages of development from seedling to maturity (7 weeks). The cellular location of the lectins differs between soybean and peanut: in soybean the lectins are generally membrane-associated, whereas in peanut plants lectin activity is present also in the soluble cytoplasmic fraction. The membrane-associated lectins appear to differ from the seed lectins of the respective plants. The function of membrane-associated lectins is discussed.Abbreviations RCA lectin of castor bean - SBA soybean agglutinin - PNA peanut agglutinin - HEPES 2-[4-(2-Hydroxyethyl)-piperazinyl-(1)]ethanesulphonic acid - MES morpholinoethane sulphonic acid - PBS phosphate-buffered saline     

Glucosylation of membrane-bound proteins by lipid-linked glucose

Particulate preparations from Pisum sativum. were able to incorporate [¹⁴C]glucose from UDP-[¹⁴C]glucose into oligosaccharide-linked lipids was formed by an oligosaccharide chain containing 7-8 glucose residues linked to dolichol, presumably via a pyrophosphate. The polymer was identified as a membrane-bound glucoprotein that could be solubilized by Triton X-100. SDS gel electrophoresis showed that a polypeptide with an apparent molecular weight of 13,000 could be glucosylated from dolichyl-phosphate-glucose. This was coincident with the electrophoretic mobility of the subunit of the pea lectin in the same system. The glucosylated protein was solubilized from the membranes by sonication and showed the same carbohydrate-binding ability as pea lectins. These results strongly suggest that pea lectins can be glucosylated by the lipid intermediate pathway.Abbreviations BSA Bovine serum albumin - Dol dolichol - SDS sodium dodecyl sulfate     

Lectin staining of the dendrite sheath in contact chemosensitive sensilla of Periplaneta americana

The two sensillum lymph cavities of contact chemosensitive sensilla of insects are separated by the dendrite sheath. The composition of the dendrite sheath in the contact chemosensitive sensilla of the ventral sensory field on the maxillary palps of Periplaneta americana L. has been studied using lectin cytochemistry. The dendrite sheaths can be isolated from the surrounding tissue and the autofluorescent cuticle by incubation of cryostat sections in phosphate-buffered saline containing 1% Triton X-100. Various lectins bind specifically to the dendrite sheath indicating the presence of sugar residues. Enzymatic digestion with hyaluronidase or trypsin prior to lectin cytochemistry decreases staining intensity, whereas treatment with neuraminidase and chitinase causes no visible change. The material of the dendrite sheath can thus be assumed to contain acid mucopolysaccharides that probably are similar to those of the receptor lymph cavities. The negatively charged, hydrophilic acid mucopolysaccharides may make the dendrite sheath permeable for small cations. These findings are discussed in the context of the functional significance of the dendrite sheath in contact chemosensory sensilla of insects.     

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.     

Characterising adhesiveness ofPhytophthora cinnamomi zoospores during encystment

During encystment,Phytophthora cinnamomi zoospores bind firmly to the host surface. We have developed a microassay to study adhesion of the zoospores to solid surfaces, both biological and non-biological. The results show that timing of the acquisition of adhesiveness during encystment correlates closely with the secretion of high molecular weight glycoproteins. The adhesive phase is short lived, occurring between 1 and 4 min after induction of encystment. During this period, cells that come into contact with a variety of surfaces (glass, plastic, and onion epidermis) become firmly attached, while cells that come into contact with one of these substrata after this period are unable to bind. Our results also show that EGTA inhibits cyst adhesion, while addition of calcium promotes cyst adhesion, especially of cysts more than 4 min old. To help identify the cyst surface component involved in adhesion we tested a number of lectins for their ability to block cyst adhesion. Soybean agglutinin andHelix pomatia agglutinin, lectins which bind to the secreted high molecular weight glycoproteins, both inhibit adhesion in the presence and absence of the hapten sugar, indicating that inhibition was non-specific. Wheatgerm agglutinin, a lectin which does not bind to the cyst surface, also blocked adhesion non-specifically.     

Immuno- and lectin-electron-microscopic investigation of the neural lobe of the hypophysis in the snake Natrix maura

Summary By the use of lectin histochemistry, and immunocytochemistry with antisera against bovine neurophysins I and II (NPs), arginine vasotocin (AVT) and mesotocin (MST), the neural lobe of the hypophysis in the snake Natrix maura was investigated at the light- and electron-microscopic levels. While paraldehyde-fuchsin stained virtually all neurosecretory endings, the periodic acid-Schiff reaction revealed only a portion of these elements. Furthermore, concanavalin-A and wheat-germ agglutinin lectins reacted with some but not all terminals. While in electron micrographs lectin-positive neurosecretory endings displayed medium-sized, pale neurosecretory granules, those from lectinnegative endings were larger and denser. The antiserum against the two NPs revealed the entire population of neurosecretory endings. The antiserum to AVT stained more numerous fiber elements than the antiserum to MST. Ultrastructurally, correlations concerning size and electron density can be found, on the one hand, between AVT-immunoreactive and lectin-positive neurosecretory granules and, on the other hand, between MST-immunoreactive and lectinnegative granules. The use of immuno-electron microscopy for the characterization of the different endings in the neural lobe and the presence of carbohydrates in some of them is discussed.This work was supported by the Directión General de Universidades e Investigación de la Junta de Andalucía (Grant BOJA 27/9/88) and the Direction General de Investigación Científica y Técnica (DGICYT Grant PB87 0710) Espaa     

Lectin activity in pollen from plants representative of thirty orders of spermatophyta

Summary Pollen extracts from a variety of species representative of thirty orders of spermatophyta, including gymnosperms, dicotyledons and monocotyledons, were examined for the presence of lectin activity by means of a hemagglutination assay. Hemagglutinating activity (HA) was detected in the pollen extracts of all the species examined, indicating that lectins are generally present in the pollen of spermatophyta. The response of this pollen hemagglutinating activity to the sugars and glycoproteins tested as potential inhibitors was identical in all species examined. Moreover, the hemagglutinating activity of pollen extracts from eight species which had been selected as representative of the gymnosperms and both subclasses of angiosperms exhibited similar properties (e.g. distribution by differential centrifugation, stability to heat, response to bivalent ions). The bulk of the hemagglutinating activity was always recovered in the pellet after centrifugation at 1000 g for 5 min. Although sequential treatments with 1% Triton X-100 and 1 M KCl were ineffective, subsequent incubation of the pellet with saline phosphate buffer released hemagglutinating activity. The solubilized hemagglutinating activity was destroyed by protease treatment, indicating that the substance(s) responsible for the activity is (are) protein in nature and, consequently, might be considered to be a lectin. The sugar specifity of the pollen lectin activity from wheat, potato and bean was compared with that of wheat germ agglutinin (WGA), potato agglutinin and bean agglutinin — the lectins present in sporophytic tissues of these plants. For all three plants, the response of the pollen lectin activity to sugars and glycoproteins was different from that shown by the lectin from sporophytic tissues.Abbreviations HA Hemagglutinating activity - PBS 150 mM Na-phosphate buffer (pH 7.2) containing 0.9% NaCl - PHA Phaseolus vulgaris agglutinin - STA Solanum tuberosum agglutinin - WGA wheat germ agglutinin     

The role of kinetosome-associated organelles in the attachment of encysting secondary zoospores ofSaprolegnia ferax to substrates

Summary Electron and fluorescence microscopy were used to identify organelles involved in attachment of secondary zoospores ofSaprolegnia ferax as they were transformed into secondary cysts. When secondary zoospores were exposed to 1.0% peptone in the absence or presence of a substrate, they began to encyst. If substrates were present when encystment was induced, the groove surface of the secondary zoospores adhered to them. The first event in attachment was secretion of contents of the kinetosome-associated organelle (K-body), which was typically oriented with the tubule-filled cavity positioned toward the cell surface of the groove region in the zoospore. The tubules which contained carbohydrates became coarsely granular, the matrix became more fibrous, and the shell remained along the membrane concavity that was formed as the K-body fused with the plasma membrane.Five minutes later, a cyst coat appeared, and cysts were not readily dislodged from a substrate. The concavity was no longer found, presumably because it had evaginated; but a layered pad of adhesion material was between the cyst coat and substrate. The layers of the adhesion pad corresponded to the structure of the matrix of K-bodies. As with the tubules of the K-body, the coarsely granular portion at the edge of the pad stained for carbohydrates. Similarly, the lectins WGA and GS-II labeled with fluorescein stained the rim of the adhesion pad on cysts, indicating the presence of glycoconjugates containing N-acetylglucosamines. Because globular areas near the kinetosomes and groove of zoospores (where K-bodies were located) also bound WGA and GS-II, K-bodies contained the same carbohydrates as the adhesion pad. We conclude that K-bodies function in the attachment of encysting zoospores to substrates as the cell differentiates. The tubular portion of the K-body matrix contains carbohydrates which might assist in the adhesion process.Abbreviations D dictyosome - EV encystment vesicle - F flagellum - C cyst - CC cyst coat - Con A concanavalin A - GS-II Griffonia simplicifolia lectin - K K-body - Kt kinetosome - M mitochondria - N nucleus - NB nuclear beak - PC peripheral cisterna - PV peripheral vesicle - S shell region of K-body matrix - SBA soybean agglutinin - R 3 anteriorly directed triplet rootlet - R 8 posteriorly directed octet rootlet - WEV water expulsion vacuole - WGA wheat germ agglutinin     

Barnacle larval destination: piloting possibilities by bacteria and lectin interaction

Modulation of metamorphosis in barnacles in response to cues of biological origin is established. The bacteria associated with the barnacles also have a role in such modulations. We isolated the bacteria, Pseudomonas aeruginosa, Bacillus pumilus and Citrobacter freundii from the shell surface of Balanus amphitrite and assayed against its cypris larvae. The former species was promotory while the latter two inhibited cyprid metamorphosis. P. aeruginosa however, when tagged with lectins specific to glucose and its derivatives, mannose and fructofuranose negated the promotory effect. Whereas, tagging of galactose derivatives translated the inhibitory effect of B. pumilus and C. freundii into a promotory one showing that lectins can alter the signals in either direction. Galactose-binding lectins have been identified in the haemolymph of barnacles, which could find their way through the excretory system to the surface. The presence of such lectins could probably provide this organism with an ability to alter the signals or cues. Microscale patchiness of bacteria is also evident on surfaces in the sea. The availability of conflicting cues in patches may help pilot the larvae to their settlement destination. Understanding these controlling mechanisms and interfering with the pathways that are involved in lectin synthesis would be a step forward in antifouling technology.     

In vivo expression and characteristics of novel alpha-D-mannose-rich glycoprotein markers of apoptotic cells

We recently established that an increased expression of alpha-D-mannose (Man)- and beta-D-galactose-rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89-95]. It was independent of cell line or apoptosis-inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-D-mannose- and beta-D-galactose-containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829-838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of alpha-Man-rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin-affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high M(W) of approximately 600 and 800 kDa) whose expressions were increased during apoptosis. Triton X-114 treatment of cell membrane samples showed that the apoptotic cell-specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI-TOF MS analysis as the microtubule-actin cross-linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G-protein beta-subunit like (Acc# BAA06185.1) and dystonin isoform beta.