Lectin-gold Conjugates Research Articles

ФУНДАМЕНТАЛЬНА ТА ПРИКЛАДНА ЛЕКТИНОЛОГІЯ В НАУКОВІЙ ДІЯЛЬНОСТІ ПРОФЕСОРА АНТОНІНИ ЯЩЕНКО (75-річному ювілею присвячується)

Professor Antonina Yashchenko belongs to the group of outstanding morphologists, who developed and continue working productively for the popularization and implementation of the fundamental aspects, as well as spreading of applied lectinology methods in the morphologic research. As the result of her numerous studies in the field of lectin histochemistry were received new data on the applicability of a set of original lectin preparations purified and characterized in the «Lectinotest» from raw materials of the Carpathian region. Lectin-peroxidase and lectin-gold conjugates were applied in immunobiological research, light and electron microscopy, the methods of indirect identification of lectin receptors were worked out. Eventually these results were collected in a DSci dissertation of A. Yashchenko «Lectins as markers in normal and pathological conditions», conferred in 2004. This dissertation included a huge amount of authorized observations concerning wide possibilities of lectin histochemistry methods for studies of cell differentiation, identification of cellular subpopulations and diagnosis of pathological processes. Professor Yashchenko intensely studied the patterns of the rearrangement and redistrubution of lectin-reactive glycopolymers in the process of embryonic and postnatal morphogenesis, found out the possibilities of lectins’ application as selective histochemical markers of certain types of cells; studied the heterogeneity of lectins binding to cellular subpopulations depending on their tissue and organ specificity, as well as on the degree of their differentiation. Based on the results of the conducted research Prefessor Yashchenko published more than 250 papers in the professional journals, as well received 2 certificates of inventions. Published in 1999 «Atlas of Microanatomy of the Oral Cavity Organs» of her authorship got «The Yaroslav Mudryy award» of the Academy of Sciences of Higher School of Ukraine. Professor Antonina Yashchenko participated as a co-author in the National textbooks «Histology, cytology, and embryology» for Medical (2018) and Dentistry (2020) students. For these, as well as other morphology-related activities, in 2021 she was granted «A honorary award of the Ukrainian scientific society of anatomists, histologists, embryologists and topographo-anatomists»

Histochemical localization of N-acetyl-galactosamine in the midgut Lutzomyia longipalpis (Diptera: Psychodidae).

The binding of lectins to the midgut of the female sand fly Lutzomyia longipalpis (Lutz & Neiva) was investigated using lectin-gold conjugates. Midguts from laboratory-reared flies provided fructose solution and/or blood fed on hamster were dissected at 6, 24, and 48 h and at 5 and 7 d after feeding. Before examination by transmission electron microscopy, each midgut was sectioned, incubated with lectins from four sources (Canavalia ensiformis [ConA], Helix pomata agglutinin [HPA], peanut agglutinin [PNA], and wheat germ agglutinin [WGA] ), then conjugated with colloidal gold. Only HPA, which is specific for N-acetyl-galactosamine (GalNAc), bound to the midgut. Binding sites were cytoplasmic secretory granules and microvilli throughout the length of the midgut epithelium. Binding occurred in sand flies fed fructose as well as in flies receiving a blood meal. The presence of GalNAc on the midgut microvilli of sand flies before, during, and after blood feeding indicates this amino-sugar is not altered by digestion. As a structural component, GalNAc may represent a terminal on a receptor molecule. The failure of the sand fly peritrophic matrix to bind WGA by N-acetylglucosamine may be caused by the complex composition of the membrane, which renders N-glycan inaccessible to the lectin-gold conjugate.

Effects of benzimidazole anthelmintics as microtubule-active drugs on the synthesis and transport of surface glycoconjugates in Hymenolepis microstoma, Echinostoma caproni, and Schistosoma mansoni.

Hymenolepis microstoma (Cestoda), Echinostoma caproni, and Schistosoma mansoni (Digenea) were exposed to benzimidazoles to determine the influence of the drugs on the secretion of glycoconjugates that protect the worms' surface. Worms were obtained from mice treated with mebendazole or albendazole, and the glycoconjugates were localized in the parasite tissues by cytochemistry using lectin-gold conjugates. Events leading to the death of H. microstoma and E. caproni extended over a medication period for at least 2-3 days, and the following interrelated phases were discernible. Upon depolymerization of the microtubules the tegumentary cytons continued to synthesize glycoconjugates for up to about 24 h. Vesicles containing the glycans accumulated in the cytons, but their microtubule-based transport to the distal tegument was inhibited. At about 1 day the Golgi complex became fragmented and the production of glycans sharply declined. As a consequence of this and an ongoing turnover of the surface coat the contents of glycoconjugates in the distal tegument decreased. Similar effects were produced by vinblastine and colchicine in vitro. In contrast, benzimidazole treatment of S. mansoni, which is reportedly inefficacious, did not alter the replenishment of the surface glycoconjugates. Diminution of the coating with glycoconjugates of the surface of drug-sensitive species constitutes a secondary effect of benzimidazoles that might, synergistically with immune mechanisms of the host, enhance the expulsion of the worms.

Glycoconjugate expression defines the origin and differentiation pathway of intestinal M-cells.

Intestinal M-cells are specialized epithelial cells located in the domes of the gut-associated lymphoid tissues, which transport antigens from the lumen to the underlying lymphoid tissue, thereby initiating immune reactions. It is assumed that M-cells arise from stem cells in the crypts, from which they migrate to the top of the domes. To study the differentiation pathway of M-cells, we used the rabbit cecal lymphoid patch in which the M-cells express high levels of alpha 1-2-linked fucose and N-acetyl-galactosamine residues in their apical membrane. Dome areas were labeled with fluorescein- and rhodamine-conjugated lectins specific for alpha 1-2-linked fucose and N-acetyl-galactosamine in vivo and in vitro, and were observed with confocal laser scanning microscopy. Ultrathin sections were double labeled with lectin-gold conjugates and the labeling density was quantified by computer-based image analysis. All cecal patch M-cells expressed alpha 1-2-linked fucose and N-acetyl-galactosamine, but the amount of the two saccharides varied considerably depending on the position of the M-cells at the base, flank, or top of the dome. In eight of 18 rabbits studied, radial strips of M-cells with common glycosylation patterns were observed, each strip associated with an individual crypt. Confocal microscopy revealed that lectin-labeled M-cells were not restricted to the dome epithelium but were also detected in the upper third of crypts surrounding the domes. The results show that M-cells are heterogeneous concerning the glycosylation pattern of membrane glycoconjugates. This pattern is modified as the M-cells differentiate and migrate from the base to the top of the dome. Radial strips of M-cells with a common proclivity of glycoconjugate expression suggest that those M-cells that derive from the same crypt have a clonal origin. The presence of (pre-) M-cells in the crypts surrounding the domes indicates that M-cells derive directly from undifferentiated crypt cells and do not develop from differentiated enterocytes.

Quantification and preservation of lectin binding by isolated cardiomyocytes

During preparation of cells for experimentation a considerable amount of bound substance is lost. Our aim was to develop a protocol which retained lectin binding to an extent similar to living cells. This procedure would use fixation procedures suited for fluorescent lectin conjugates and gold-conjugates to be visualized by light- and electron microscopy, respectively. We tested glutaraldehyde and paraformaldehyde in different concentrations before and after lectin binding, different buffers and divalent cations, as additives, to determine the effects on preservation of lectin binding. Lectin binding was visualized and semiquantitatively evaluated by image analysis in the light microscope after silver enhancement of lectin-gold conjugates and by using tetramethyl rhodaminyl isothiocyanate (TRITC)-conjugated lectins. Preservation of lectin binding was best visualized with fluorescent lectin conjugates, whereas during silver enhancement procedures of gold-conjugated lectins, a considerable amount of bound lectins was lost. In general, lectin binding to living cells followed by fixation is superior to fixation before lectin binding. Unfavourable combinations of fixatives and buffers can cause a loss of more than 90% bound lectin. In our experiments with freshly isolated guinea pig cardiomyocytes, lectin binding was best when we used Na-cacodylate buffer with glutaraldehyde fixation (0.1%) after binding of lectins to the living cells.

Basement membrane lectin binding sites are decreased in the esophageal endoderm during the arrival of presumptive muscle mesenchyme in the developing asteroid Pisaster ochraceus.

Basement membranes (BMs) of vertebrates and invertebrates have been shown to contain glycoproteins and proteoglycans, which include oligosaccharides and glycosaminoglycans. Lectin binding sites were characterized in the BM of gastrulating embryos of the starfish, Pisaster ochraceus. In early and mid-gastrulae, the fluorescein isothiocyanate (FITC)-lectin conjugates of concanavalin A (Con A) and wheat germ agglutinin (WGA) reveal the presence of mannose/glucose and glucosamine/sialic acid residues in the BM of all regions of the embryos. However, in the late gastrula embryo, an apparent reduction of these components is observed over the esophageal BM. Ultrastructural studies using the lectin-gold conjugates Con A, Limax flavus agglutinin (LFA), specific for sialic acid, and Dolichos biflorus agglutinin (DBA), specific for galactosamine, demonstrate that most mannose/glucose and galactosamine-containing residues lie in the lamina densa, whereas most sialic acid residues are located over the lamina lucida. In addition, a statistical analysis of lectin binding in the late gastrula embryo reveals that the amount of labelling with both Con A and LFA is significantly reduced in the esophageal region, suggesting that mannose/glucose and sialic acid residues are reduced in this region. These results confirm the observations of the FITC-lectin studies described above. They also confirm earlier studies that demonstrated a difference in BM morphology of the esophageal region (Crawford, '88). Mesenchyme cells, some of which arise from the forming coeloms (Crawford, '90), and which may represent a distinct population, colonize exclusively on this esophageal BM, where they later differentiate into muscle. Quantitative differences in BM glycoconjugates may act to direct the presumptive muscle cells to the region of the esophagus.

Formation, properties and degradation of the peritrophic membranes of larval and adult fleshflies,Parasarcophaga argyrostoma (Insecta, Diptera)

Parasarcophaga argyrostoma larvae continuously secrete a single, tube-like peritrophic membrane (PM), which has an electron-dense layer on the lumen side and a thicker chitin-containing electron-lucent part on the epithelium side. In the adult fleshfly, the secretion of PMs starts immediately after emergence. The initial part of the PMs is twisted and tight. The formation zone is folded with two separate secretory pads in which two tube-like PMs are formed continuously. The PMs are different, morphologically and with respect to their peripheral carbohydrate residues. The latter could be demonstrated with lectin gold conjugates. PM 1 consists of an electron-dense, chitin-free layer on the lumen side and a thicker part which contains chitin microfibrils in the matrix. PM 2 appears fluffy and has chitin microfibrils in its matrix, too. Chitin could be localized with WGA gold. Incubation of isolated PM 1 with lectin gold resulted in a peculiar pattern of bound lectins and gaps on the electron dense layer which otherwise appeared to be homogenous. Degradation of peritrophic membranes takes place in the hindgut. The cuticle of the anterior hindgut is studded with small teeth, which seem to be responsible for mechanical degradation of the peritrophic membranes into frayed pieces. This may be completed by the teeth on the rectal pads. From the appearance of the remnants of the peritrophic membranes it can be inferred that chemical degradation takes place in the hindgut.

Lectin histochemistry of the hyaline layer in the asteroid, pisaster ochraceus

Six different lectins were used to study the carbohydrate nature of the hyaline layer (HL), the external extracellular matrix of the starfish embryo. Thin sections of embryos fixed in the late gastrula stage were incubated with five fluoresceinated lectins: Con A, WGA, RCA, UEA-I, and SBA. All but UEA-I labelled the HL, suggesting that the following sugars are present: mannose and/or glucose, glcNAc and/or Neu5Ac, galactose, and galNAc. The different lectins produced variable degrees of labelling, with WGA, RCA, and SBA producing more intense labelling than Con A. Binding of lectins by the HL was studied at the ultrastructural level by exposing ultrathin sections to the following lectin-gold conjugates: Con A, WGA, PNA, SBA, and LFA. Lectin binding was observed over the various regions of the HL, recognized by Crawford and Abed (J. Morphol. 176:235-246, '86), i.e., the intervillus layer, the supporting layer and the coarse outer meshwork. Local differences in labelling patterns were observed among the various lectins, with SBA labelling all regions intensely, WGA and PNA labelling the supporting layer predominantly, and Con A labelling the HL only lightly. No labelling was observed with LFA. These lectin-labelling patterns in the HL demonstrate the presence of different glycoconjugates in different regions of the HL, suggesting that the layers differ biochemically. The existence of biochemical differences strengthens the idea that each layer may have different functions in the developing starfish embryo.

Binding of lectin-gold conjugates by two Trypanosoma cruzi strains in ampullae and rectum of Triatoma infestans

The ampullae and recta of Triatoma infestans infected with Trypanosoma cruzi were incubated with lectin-gold conjugates of wheat germ agglutinin (WGA) and soy bean agglutinin (SBA) and investigated by electron microscopy. T. cruzi colonized the whole lumen of the ampullae. The rectal pads with their intensive intracellular membrane system and thin cuticle were often covered by a compact layer of flagellates, whereas the remaining rectal sac - recognizable by a thick layered cuticle - was colonized less densely. All freely accessible stages of T. cruzi strain ‘Chile 5’ (Zymodeme 1) showed a reaction with SBA-gold complexes but not with WGA, revealing that N-acetyl-galactosamine and/or galactose but no N-acetyl-glucosamine residues were present on the surface. T. cruzi strain ‘Chile 7’ (Zymodeme 2) also reacted with SBA. WGA-bovine serum albumin-gold conjugates bound strongly to the surface of epimastigotes of this strain, in variable amounts to stages in transition to trypomastigotes, but not to metacyclic trypomastigotes.

Gold-conjugated arabinogalactan-protein and other lectins as ultrastructural probes for the wheat/stem rust complex

Arabinogalactan-protein (AGP, "beta-lectin") was isolated from leek seeds, tested for specificity, conjugated with gold colloids, and used as a cytochemical probe to detect beta-linked bound sugars in ultrathin sections of wheat leaves infected with a compatible race of stem rust fungus. Similar sections were probed with other gold-labeled lectins to detect specific sugars. AGP-gold detected beta-glycosyl in all fungal walls and in the extrahaustorial matrix. Other lectin gold conjugates localized galactose in all fungal walls except in walls of the haustorial body. Limulus polyphemus lectin bound only to the outermost layer of intercellular hyphal walls of the fungus. Binding of these lectins was inhibited by their appropriate haptens and was diminished or abolished in specimens pretreated with protease, indicating that the target substances in the tissue were proteinaceous or that polysaccharides possessing affinity to the lectin probes had been removed by the enzyme from a proteinaceous matrix by passive escape. Binding of Lotus tetragonolobus lectin was limited to the two outermost fungal wall layers but was not hapten-inhibitable. Limax flavus lectin, specific for sialic acids, had no affinity to any structure in the sections. In the fungus, the most complex structure was the outermost wall layer of intercellular hyphal cells; it had affinity to all lectins tried so far, except to Limax flavus lectin and to wheat germ lectin included in an earlier study. In the host, AGP and the galactose-specific lectins bound to the inner domain of the wall in areas not in contact with the fungus. At host cell penetration sites, affinity to these lectins often extended throughout the host wall, confirming that it is modified at these sites. Pre-treatment with protease had no effect on lectin binding to the host wall. After protease treatment, host starch granules retained affinity to galactose-specific lectins, but lost affinity for AGP.

Differences in binding of lectin-gold conjugates by Trypanosoma cruzi and Blastocrithidia triatomae (Trypanosomatidae) in the intestine of Triatoma infestans (Reduviidae).

Two species of trypanosomatid flagellates, Trypanosoma cruzi and Blastocrithidia triatomae which develop in the intestine of Triatoma infestans, were incubated with lectin-gold conjugates to demonstrate sugar components of the cell surface with the electron microscope. T. cruzi, strain "Chile 5" (zymodeme 1), bound SBA-gold, revealing N-acetyl-galactosamine and/or galactose residues. WGA-BSA-gold, specific for N-acetylglucosamine, did not bind to the surface of this flagellate. On the other hand, N-acetyl-glucosamine bound WGA-BSA-gold on the cell membrane of epimastigotes and cysts of B. triatomae, whereas SBA-gold was not bound by flagellates which were attached to the midgut epithelium and the rectum cuticle. Among other criteria, these differences in lectin binding may facilitate the discrimination of the two parasite species in the vector of Chagas' disease.