Light Microscopic Lectin Histochemistry on Celloidin Stabilized Cryostat Sections of Rat Colon

Celloidin stabilized pre-epithelial mucus gel (PMG) is stained in cryostat sections of the rat colon for light microscopic lectin histochemistry. Specific sugar residues of the mucins are demonstrated both in the PMG and in the mucin-containing cells of the mucosa.     

Light Microscopic Lectin Histochemistry on Celloidin Stabilized Cryostat Sections of Rat Colon

Celloidin stabilized pre-epithelial mucus gel (PMG) is stained in cryostat sections of the rat colon for light microscopic lectin histochemistry. Specific sugar residues of the mucins are demonstrated both in the PMG and in the mucin-containing cells of the mucosa.     

Calcium amelioration of cadmium-induced cytotoxicity in cultured rat hepatocytes

Summary Parenchymal hepatocytes from neonatal rats were isolated, cultured about 24 h, exposed to cadmium with or without calcium, and processed for scanning electron microscopy. To assess the severity of cadmium-induced changes, exposed hepatocytes were categorized based upon the extent of morphological damage. Differences in surface blebbing, alterations in microvilli, variations in the degree of swelling, and changes in cell shape were used to categorize the severity of cell damage. A double-blind morphometric analysis (a geometricostatistical processing of two-dimensional data for the collection of three-dimensional information) of cellular changes was conducted for each exposure time and for each concentration of cadmium in the presence or absence of calcium. Significant decreases occurred in the percent relative volume of normal, flattened cells present in cultures exposed for 30 min to 50 or 100 μM cadmium in the absence of calcium. In contrast, the percent relative volume of severely damaged spherical cells was significantly increased after exposure to solutions containing 50 or 100 μM cadmium and lacking calcium. Percent relative volume of intermediate cells (which were slightly swollen and showed changes in microvillar number) was significantly increased following a 30 min exposure to all cadmium concentrations in the absence of calcium. The examination of hepatocytes exposed for 60 min showed that the degree of cadmium-induced cytotoxicity was more severe in the absence of calcium than was the case for the hepatocyte cultures exposed for 30 min: approximately 30% more spherical cells and 30% fewer flattened cells were present if cultures were exposed in the absence of calcium for 60 min compared to those exposed for 30 min. The degree of blebbing was significantly greater at all cadmium concentrations in the absence of calcium. The presence of calcium, therefore, reduced cadmium-induced cytotoxicity in primary cultures of rat hepatocytes subjected to morphometric analysis after scanning electron microscopy.     

Lectin histochemistry of the human fetal subcommissural organ

The subcommissural organ (SCO) of 7 human fetuses, 3 to 6.5 months old, was investigated by means of: (i) immunocytochemistry employing three different antisera against secretory products extracted from the bovine SCO and Reissner's fiber; (ii) lectin binding using concanavalin A (Con A; affinity: mannose, glucose), wheat-germ agglutinin (WGA; affinity: N-acetyl-glucosamine, sialic acid), and Limax flavus agglutinin (LFA; affinity: sialic acid). Sections of bovine SCO were processed simultaneously and examined for comparative purposes. The human fetal SCO displayed lectin-binding properties identical to those in the SCO of other mammals. Thus, Con A-binding sites were restricted to abundant supranuclear structures that most likely corresponded to the rough endoplasmic reticulum, but were missing from granules located in the apical cytoplasm. The latter secretory material was strongly WGA- and LFA-positive and formed a distinct zone in the most apical portion of the ependymal cells. In contrast, this type of reactivity was missing in the adjacent cells of ependyma proper. In the bovine SCO, LFA-positive granules were also aggregated in an apical layer. The secretory material in the bovine SCO, especially its apical granular component, was strongly immunoreactive with the three antisera used; the human fetal SCO, however, lacked this immunoreactivity. It is postulated that the SCO of human fetuses secretes glycoproteins with a carbohydrate chain similar to--and a protein backbone different from--the secretions elaborated by the SCO of other vertebrate species.     

Seasonal pattern of glycosylation in frog liver

The circannual behaviour of glycosylation and protein synthesis in frog liver slices was studied following the incorporation of³H-galactose and¹⁴C-glucosamine into glycolipids and glycoproteins and³H-leucine into proteins. The activity of two enzymes the galactosyl-transferase and the N-acetyl-glucosaminyl-1-P-transferase was determined. The incorporations of both sugars into the soluble fraction and into the lipid extract present a maximum during the spring-summer period. The incorporation into the protein fraction displays a different pattern:¹⁴C-Glucosamine and³H-leucine incorporation increases from winter to a maximum in autumn; the incorporation of³H-Galactose has a sharp peak during spring. The pattern of glycosyltransferase activities is similar to the pattern of incorporation of the two saccharides into proteins, indicating these enzymes as important control points for glycosylation in Anurae.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Differences in the glycosylation of rat submandibular kallikreins

The glycosylations of five different rat submandibular kallikreins, rK1, rK2, rK7, rK9 and rK10, vacuum-blotted onto nitrocellulose membranes, have been studied by means of labelled lectins using enhanced chemiluminescence detection. The results demonstrated that individual submandibular kallikreins are not heavily glycosylated in rats, but consistently show different patterns of glycosylation. Following digestion of slot-blotted enzymes with peptide-N-glycosidase F (PNGase): binding by lectin fromLens culinaris (Man-directed) was abolished, whilst that of lectin fromMaclura pomifera (Gal1,3GalNAc-directed) persisted (but could be abolished by periodate oxidation and endo--N-acetylgalactosaminidase digestion), revealing that there are O- as well as N-linked sugar chains on the kallikreins; a novel observation for this family of enzymes. The presence of GalNAc in addition to GlcNAc, Fuc, Gal and Man, in sugar chains of rK1 was confirmed by high pH anion exchange chromatography following acid hydrolysis. Different intensities of binding by lectin fromLimax flavus (NeuNAc-directed) suggest that sialylation of individual kallikreins differs, whilst sialidase and PNGase digestions suggest that sialic acid is the terminal residue of some N-linked but not O-linked structures.     

Characterization of sugar moieties and oligosaccharide sequences in the distal intestinal epithelium of the rainbow trout by means of lectin histochemistry

Lectin histochemical study was performed on the distal intestinal mucosa of the rainbow trout. A panel of 25 biotinylated leetins for detection of sugar residues and oligosaccharide sequences of glycoconjugates on Bouin's solution-fixed paraffin embedded sections were used as probes. The avidin-biotin-peroxidase complex (ABC) was used to visualize the binding sites of lectins. A rich supply of carbohydrates was detected in this gut mucosal region. Marked differences in lectin labelling of goblet cells and enterocytes (absorptive cells) probably reflect various functional activities. The results are compared with those (scarce) obtained from other teleosts. Species differences in carbohydrate residue composition may be related to various feeding habits. The roles of posterior intestinal glycoconjugates in host-parasite interaction, macromolecular ingestion and immunological implications are discussed.     

The effects of alcohol on rat placenta

In this study, daily food and water consumption and body weights, histopathology of placenta, tenascin (TN), type IV collagen and EGF and its receptor immunolocalization in the placenta of albino rats treated with two doses of alcohol (1 and 5 g kg(-1) day(-1)) were determined. Alcohol was administered in three different periods i.e. the whole 4 weeks before the pregnancy, during the pregnancy, and during the 4 weeks before the pregnancy plus pregnancy itself. The samples of placenta obtained from control and treated rats on days 10, 12, 14, 16, 18, 20 and 21 of gestation were evaluated morphologically and fixed for histology and immunohistochemistry. Some differences in food and water consumption between the groups were determined. The placental weight, especially in the groups receiving 1 and 5 g kg(-1) day(-1) alcohol during the pregnancy, showed increases. The changes in placental histology such as increases in the number and the size of trophoblastic giant cells, cytoplasmic dissolution and nuclear polymorphism, degenerations in spongiotrophoblasts, hyperemia at the basal zone and labyrinth, hyperplasia at the labyrinth and irregular vascularization were seen particularly in the groups receiving alcohol during the pregnancy, and during the 4 weeks before the pregnancy plus pregnancy itself. Increases in the immunolocalization of TN and type IV collagen and decreases in the immunolocalization of EGF and EGFR in the placentas of alcohol-receiving rats were found. In conclusion, ethanol treatment during pregnancy in rats affected placentation and the immunolocalization of TN, type IV collagen, EGF and EGFR in the placentas.     

Lectin histochemical localization of N- and O-linked oligosaccharides during the spermiogenesis of the urodele amphibian Pleurodeles waltl

The aim of this work is the characterization of the glycoconjugates of the spermatids during the spermiogenesis of the testis of an urodele amphibian, Pleurodeles waltl, by means of lectins in combination with several chemical and enzymatic procedures, in order to establish the distribution of N- and O-linked oligosaccharides in these cells. The acrosome was the most relevant lectin-labeled structure. The O-linked oligosaccharides contained DBA- and SBA-positive GalNAc, AAA-positive Fuc and PNA-positive Gal1,3GalNAc. Sialic acid was scarcely observed, the Neu5Ac2,-3Gal1,4GlcNAc sequence was found in N-linked oligosaccharides. Additionally, N-linked oligosaccharides containing HPA-positive GalNAc and AAA-positive Fuc were found. Moreover, with some lectins the acrosome showed a variable composition of the oligosaccharides in the different steps of the sperm maturation. Some residues were found only in the early steps in maturating acrosome, while others were in the later steps, showing that acrosomal glycoconjugates are modified during acrosome development in spermiogenesis. The changes observed during acrosome maturation suggest the existence of a predetermined pattern of storage of the acrosome components and a progressive compression of them.     

Expression patterns of pituitary adenylate cyclase activating polypeptide and its type I receptor mRNAs in the rat placenta

Pituitary adenylate cyclase activating polypeptide (PACAP) was first isolated from ovine hypothalamus and is known to act as a tropic factor in various cells. Recent report revealed the expression of PACAP and the PACAP type I (PAC(1)) receptor in human and rat placentas at term. Placenta is a critical organ that synthesizes several growth and angiogenic factors for its own growth as well as fetal development. However, there is little information regarding the expression pattern and cellular localization of PACAP and PAC(1) during pregnancy. The aim of this study was to define the expression and distribution of PACAP and PAC(1) receptor mRNAs in the rat placenta during pregnancy. PACAP and PAC(1) receptor mRNAs were expressed in decidual cells, chorionic vessels, and stromal cells of the chorionic villi. Interestingly, the expression of these genes varied with the day of gestation. For example, PACAP and PAC(1) receptor mRNAs expressed in decidual cells on day 13.5 and 15.5, their expression was strong in chorionic vessels and stromal cells of the chorionic villi within the labyrinth zone on day 17.5, 19.5, and 21.5. In fact, as gestation advanced, the expression of PACAP and PAC(1) receptor mRNAs in the decidua cells disappeared, as their high expression became evident in the chorionic vessels and stromal cells of the chorionic villi. Our finding that PACAP and the PAC(1) receptor are co-localized and their genes seemingly co-regulated within specific placental areas, strongly suggest that this peptide may play an important role, as an autoregulator or pararegulator via its PAC(1) receptor, in physiological functioning of the placenta for gestational maintenance.     

Some lectin binding properties of the tongue of the mole rat,Nannospalax xanthodon

We investigated carbohydrate residues on the epithelial surface, in the epithelial cells and in gland cells of the tongue of the mole rat using histochemical methods. We used horseradish peroxidase-conjugated lectins from Helix pomatia (HPA), Arachishypogaea (PNA), Ulexeuropaeus (UEA I), Canavaliaensiformis (Con A). The most intense reactivity was observed in the keratin layer with HPA, UEA I and Con A, and in the epithelial cells with UEA I and Con A. In the glands, we found strong reactivity in serous cells with HPA and Con A, and in mucous cells with HPA and UEA I. PNA did not bind to epithelial or gland cells. Consequently, GlcNAc, fucose and α-D-mannose terminal glycoconjugates are distributed widely; GalNAc terminal glycoconjugates appeared in small amounts.     

Lectin histochemistry for in situ profiling of rat colon sialoglycoconjugates

The growing interest in glycoconjugates expressed and released by the epithelium of the intestinal mucosa is tightly related to the multiple functional roles attributed to sialic acid and its derivatives. In the present work, biotin and HRP conjugated lectins were used to detect the sialylation pattern and to identify specific structural features of sialoderivatives in the rat colon. In particular, the occurrence and distribution of sialic acids linked alpha2,6 to D-Gal/D-GalNAc and alpha2,3 to D-Gal were directly demonstrated with SNA and MAL II binding, respectively. In addition, in order to by-pass the specificity problems of SNA and MAL II as histochemical reagents, as well as to look for additional and complementary information about acetylation degree and sites, we combined sialidase digestion, potassium hydroxide deacetylation, and differential periodate oxidation with PNA and DBA binding. The data showed the distribution and structure of sialic acid-beta-D-Gal(1-3)-D-GalNAc and sialic acid-D-GalNac sequences, which proved to be widely distributed as cellular components or secretory products in surface goblet cells and crypt cells of the colonic epithelium. A high degree of O-acetylation, with acetyl groups mainly at 9 and 4 positions, was found, showing an increasing gradient from the proximal to distal portion of the colon. These results, which largely reproduce the sialylation pattern in other species, contribute new insights in defining the tissue specific expression of sialoderivatives in the colonic mucosa, and testify to their high heterogeneity which the wide range of sialic acid functional correlates in the intestinal tract depend on.     

Oxytocinase-immunohistochemical demonstration in the immature and term human placenta

Summary Oxytocinase (cystine aminopeptidase) was purified from human retroplacental serum by a combination of fractional precipitation, hydroxylapatite chromatography and gel exlusion chromatography on Sephadex G-200. The purified enzyme possessed a specific activity of 980 mIU/mg using L-cystine-di-p-nitroanilide as substrate. This represented a 3200 fold concentration from the starting material in an overall yield of 12%. Antibodies against oxytocinase were raised in rabbits and the -globulin fraction labelled with fluorescein isothiocyanate prior to its use in the immunofluorescence histochemical localization of the enzyme in human placental tissue. Oxytocinase was confined to the syncytiotrophoblastic cells of normal term, and immature placentas as well as in placentas from patients suffering from severe toxaemia. Specific immunofluorescence was also present in the outer margins of the chorion and to a lesser extent in the amnion.This work was financed by a grant from The Medical Research Council of New Zealand.     

Ultrastructure and histochemistry of rat myocardial capillary endothelial cells in response to diabetes and hypertension

INTRODUCTIONThe streptozotocin [STZ]-induced diabetes and NO-deficient hypertension are widely used experimental modelsfor the investigation of heart failure [1-4]. Cardiac dys-function in both models is consequence of different sig-nal and metabolic dera…     

Alteration of the sialylation pattern of the murine tooth germ after ethanol exposure

BACKGROUND: Ethanol consumption during pregnancy leads to changes in murine dental morphogenesis, dental size, cellular differentiation, enamel mineralization, and delayed eruption. It has been proposed that glycoproteins play a role during embryonic dental development that may determine the dental morphological pattern and extracellular matrix secretion. O-glycosylation and sialylation appear to actively participate in the differentiation and maturation processes. Because glycosylation may be affected by teratogens that can alter the maturation of several organisms, in this work we describe the main modifications of the sialylation pattern in prenatal day (PD) 18.5 murine tooth germs exposed to ethanol. METHODS: Pregnant female mice were divided into groups that were given 15% or 20% ethanol solutions, or water as a control. The histochemistry of tooth germs from PD 18.5 fetuses was revealed with lectins specific for sialic acid (Neu5Ac), such as Sambucus nigra (SNA), Maackia amurensis (MAA), and Machrobrachium rosenbergii (MRL), and for sialylated-O-glycosidically linked glycans, such as Amaranthus leucocarpus (ALL). RESULTS: The basement membrane, preameloblasts, inner-enamel epithelium, preodontoblasts, and subodontoblastic cells of the test groups showed changes in labeling according to the 4 lectins used. Intranuclear staining was observed with SNA (specific for Neu5Acalpha2,6Gal/GalNAc) in the control group, but this was reduced in the test groups. The nuclei of dental papillary cells under the experimental conditions were stained with MAA (Neu5Acalpha2,3Gal). CONCLUSIONS: Dental development involves different types of sialylated O-glycosidically linked glycans that are likely to regulate cell-to-cell and cell-to-matrix interactions. Our results suggest that ethanol consumption during pregnancy alters the sialylation pattern during murine dental morphogenesis.     

Endopolyploid and proliferating trophoblast cells express different patterns of intracellular cytokeratin and glycogen localization in the rat placenta

The presence of keratin intermediate filaments is a characteristic of trophoblast differentiation. Meantime, their intracellular localization in the functionally different subtypes of placental trophoblast is poorly investigated in rodent, whereas their placentae are being broadly investigated in recent years as a model of the feto-maternal interaction. The purpose was to study the intracellular distribution of cytokeratin filaments in correlation with glycogen deposits, both being important constituents of the trophoblast cells in rat placenta. Different rat trophoblast cell populations exhibited different patterns of cytokeratin immunolocalization. The most intensive immunostaining was observed in the highly endopolyploid SGTCs (secondary giant trophoblast cells) at the border with decidua basalis. The most prominent cytokeratin-positive threads were found at the periphery of cytoplasm and in the extensive system of cytoplasmic sprouts by which the SGTC connect each other. Similar cytokeratin intensity and distribution was detected in the TSC (trabecular spongiotrophoblast cells) of the junctional zone of placenta that line the lacunae with the maternal blood. Clusters of highly proliferative pre-glycogen as well as glycogen cells showed some weaker cytokeratin signals mostly in the perinuclear and peripheral zones of cytoplasm. At the 11.5th to the 13.5th day of gestation, the interstitial and endovascular invasive endopolyploid TGTCs (tertiary giant trophoblast cells) prove the intensive cytokeratin staining throughout the cytoplasm and its sprouts. Meantime, the TGTCs were glycogen negative. By contrast, glycogen was heavily accumulated in the glycogen cells that belong both to the junctional zone of placenta and the cuff of the central arterial channel underlying the monolayer of endovascularly invading TGTCs. Thus, the TGTCs that are first to penetrate into the depth of the uterine wall do not contain glycogen but are accompanied by the glycogen-rich cells. The SGTC also contained the prominent deposits of glycogen at the periphery of cytoplasm and in the cytoplasmic sprouts. At the 16th day of gestation, an extensive interstitial invasion of the cytokeratin-positive glycogen trophoblast cells from the junctional zone was observed. The patterns of cytokeratin and glycogen intracellular localization are specific for each subtype of the rat trophoblast; that is, most probably, accounted for by the functional diversity of different trophoblast populations, i.e. patterns of invasion/phagocytosis and their involvement in a barrier at the feto-maternal interface.     

Lectin histochemistry of human placenta in the normal state and in uterine inertia]

The lectin-peroxidase technique has been used. The peanut lectin is able to label selectively Kashchenko--Hofbauer cells, villi of the chorion; the soya lectin--decidual cells in the maternal part of the placenta. Prolonged labour development is connected with disappearance of Kashchenko--Hofbauer cells, with a reduced binding of the Laburnum anagyroides lectin in combination with an intensified peanut lectin and concanavalin A binding with glycopolymeres of the villi of the chorion.     

Distribution of lectin-binding glycosidic residues in the hamster follicular oocytes and their modifications in the zona pellucida after ovulation.

In the present study, we have employed a battery of colloidal gold-tagged lectins as probes in conjunction with quantitative analysis to demonstrate the distribution and changes of carbohydrate residues in the hamster zona pellucida (ZP) during ovarian follicular development and during transit of the oocyte through the oviduct after ovulation. High-resolution lectin-gold cytochemistry performed on thin sections of LR White-embedded ovaries revealed a moderate to strong reactivity to WGA, PNA, DSA, AAA, and MAA over the entire thickness of the ZP of ovarian oocytes at different stages of follicular development. Labeling intensity over the ZP progressively increased as follicles matured in the ovary. In parallel, there was an association of labeling by gold particles with cortical granules, stacks of Golgi saccules, and complex structures called vesicular aggregates in the oocyte proper especially during the late stages of follicular growth. In contrary, labeling with each of HPA, DBA, and BSAIB(4) was absent in the ovary but was found to be localized over Golgi complexes and secretory granules in the non-ciliated secretory cells of the oviduct. When ovulated oocytes were labeled with each of HPA, WGA, RCA-I, PNA, DSA, BSAIB(4), AAA, MAA, and DBA, the ZP and several organelles in the oocyte proper presented a differential distribution of lectin-binding sites. Quantitative analysis was also performed on labeling by lectin-gold complexes that bind specifically to the ZP of mature follicular and ovulated oocytes. Quantitative evaluation revealed heterogeneous labeling between the inner and the outer zone of the ZP. A significant increase in the labeling densities in both inner and outer ZP was noted when tissue sections of ovulated oocytes were labeled with RCA-I or AAA. Tissue sections of ovaries labeled with WGA demonstrated a significant increase in the density of labeling in the outer layer of the ZP. Labeling by PNA, DSA, and MAA, however, showed a significant decrease in both the inner and outer portions of the ZP. Together, these results suggest that in the hamster, glycoproteins carrying specific sugar residues are added to the ZP of ovarian follicles during the early stages of folliculogenesis and are processed through a common secretory machinery, and that there is a significant change in both the sugar moieties and distribution of glycoproteins in the ZP following ovulation. Our results also showed that the hamster oviduct plays an important role in contributing certain glycoproteins to the ZP suggesting that the sugar moieties of these oviductal glycoproteins may have functional significance in fertilization.