Dolichos Biflorus Agglutinin Binding Research Articles

Phosphorylcholine esterase is critical for Dolichos biflorus and Helix pomatia agglutinin binding to pneumococcal teichoic acid.

Streptococcus pneumoniae (the pneumococcus) has wall teichoic acid (WTA) and lipoteichoic acid (LTA) expressing the Forssman antigen (FA). Two lectins, Dolichos biflorus agglutinin (DBA) and Helix pomatia agglutinin (HPA), are known to bind FA. To determine the molecular structure targeted by these two lectins, different pneumococcal strains were studied for DBA/HPA binding with flow cytometry and fluorescence microscopy. Genetic experiments were used to further examine the lectins' molecular target. Twelve strains were positive for DBA binding, whereas three were negative. Super-resolution microscopy showed that DBA stained only the subcapsular area of pneumococci. The three DBA nonbinders showed no phosphorylcholine esterase (Pce) activity in vitro, whereas 10 DBA binders displayed Pce activity (the remaining two strains were DBA binders with no Pce activity in vitro). The pcegene sequence for 10 representative strains revealed two functional pce alleles, the previously recognized "allele A" and a newly discovered "allele B" (with 12 additional nucleotides). Isolates with allele B showed no Pce activity in vitro but did bind to DBA, indicating allele B Pce is functional in vivo. Genetic transfer experiments confirmed that either allele is sufficient (and necessary) for DBA binding. The three DBA nonbinders had various mutations that affected Pce function. Observations with HPA were identical to those with DBA. We show that DBA and HPA bind only to the WTA/LTA of pneumococcal isolates with a functional Pce enzyme. A newly discovered Pce variant (allele B) is functional in vivo but nonfunctional when assayed in vitro.

B4GALNT2 and xenotransplantation: A newly appreciated xenogeneic antigen.

Analysis of non‐Gal antibody induced after pig‐to‐baboon cardiac xenotransplantation identified the glycan produced by porcine beta‐1,4‐N‐acetyl‐galactosaminyltransferase 2 (B4GALNT2) as an immunogenic xenotransplantation antigen. The porcine B4GALNT2 enzyme is homologous to the human enzyme, which synthesizes the human SDa blood group antigen. Most humans produce low levels of anti‐SDa IgM which polyagglutinates red blood cells from rare individuals with high levels of SDa expression. The SDa glycan is also present on GM2 gangliosides. Clinical GM2 vaccination studies for melanoma patients suggest that a human antibody response to SDa can be induced. Expression of porcine B4GALNT2 in human HEK293 cells results in increased binding of anti‐SDa antibody and increased binding of Dolichos biflorus agglutinin (DBA), a lectin commonly used to detect SDa. In pigs, B4GALNT2 is expressed by vascular endothelial cells and endothelial cells from a wide variety of pig backgrounds stain with DBA, suggesting that porcine vascular expression of B4GALNT2 is not polymorphic. Mutations in B4GALNT2 have been engineered in mice and pigs. In both species, the B4GALNT2‐KO animals are apparently normal and no longer show evidence of SDa antigen expression. Pig tissues with a mutation in B4GALNT2, added to a background of alpha‐1,3‐galactosyltransferase deficient (GGTA1‐KO) and cytidine monophosphate‐N‐acetylneuraminic acid hydroxylase deficient (CMAH‐KO), show reduced antibody binding, confirming the presence of B4GALNT2‐dependent antibodies in both humans and non‐human primates. Preclinical xenotransplantation using B4GALNT2‐deficient donors has recently been reported. Elimination of this source of immunogenic pig antigen should minimize acute injury by preformed anti‐pig antibody and eliminate an induced clinical immune response to this newly appreciated xenotransplantation antigen.

Glycosylation and immunocytochemistry of binucleate cells in pronghorn (Antilocapra americana, Antilocapridae) show features of both Giraffidae and Bovidae

Although the pronghorn (Antilocapra americana) resembles an antelope, its nearest relatives are the giraffe and okapi. In this study we have examined the placentae of 6 pronghorns using lectin- and immunocytochemistry to identify giraffid and bovid features. Binucleate cells (BNC) of the placenta exhibited features intermediate between those of the giraffe and bovine; Dolichos biflorus agglutinin binding – strong in the bovine BNC and absent in the giraffe – was evident in only a subpopulation of BNC while binding to blood vessels, as in the giraffe. Binding of Phytolacca americana agglutinin resembled that of the giraffe and okapi whereas many other glycans were found in all four clades. PAG antigens were similar to bovine and okapi but not giraffe. In summary, although the pronghorn outwardly resembles an antelope, placental BNC show giraffid features. Although each clade has its own individual characteristics, there are far more similarities than differences between them, emphasizing the common ancestry of all four clades.

Complex Carbohydrate Marker to Distinguish Mucins from Low Grade Neoplasms versus Adenocarcinomas in the Colon Causing Pseudomyxoma Peritonei

Introduction: Normal colonic epithelial cells produce mucins which are more than 50% carbohydrate and have characteristic terminal sugars on the oligosaccharide chains that can be recognized by lectins. A goblet cell in the upper portion of the colonic crypt makes mucin with a terminal sugar which binds the lectins Dolichos biflorus agglutinin (DBA) and soybean agglutinin (SBA). The lower portion of the crypt has mucin with a terminal sugar residue which binds to Ricinus communis agglutinin-1 (RCA-1). Colon cancer produces mucin that binds peanut agglutinin (PNA). The goal of this study was to use lectin binding patterns in order to differentiate mucins secreted by low grade neoplasms from mucins produced by adenocarcinomas in the colon causing pseudomyxoma peritonei. Methods: Normal and diseased colon specimens were obtained from 31 patients with mucinous lesions and prepared for fluorescent lectin microscopy using fluorescein isothicyanate (FITC)-conjugated lectins: DBA, SBA, RCA-1 and PNA. The FITC-lectin labeling and pathological interpretations were performed independently. Results: 23/23 of the adenocarcinoma cases were positive for FITC PNA labeling versus 3/7 of the low grade neoplasm cases, a difference that was statistically significant (p=0.001 by chi-square). Conclusion: PMP is a disease characterized by a mucinous fluid collection in the abdomen and pelvis caused by mucus-producing adenomas or adenocarcinomas. The disease can behave very aggressively clinically. In this study, PNA binds to the mucins in all tumor sections that are adenocarcinomas. It was expected that SBA and DBA would bind similarly to the same tumor section but that was not always the case. This might be due to subtle differences in what these two GalNAc-binding lectins recognize and has an undetermined significance. It is reasonable to theorize that a tumor section with strong PNA binding and weaker SBA, DBA, or RCA-1 binding could represent increased dysplasia within the tumor and it will be interesting to compare this data with clinical outcomes. Further work will include more cases of low grade neoplasms as well as long-term clinical outcomes in order to strengthen the hypothesis that the absence of PNA binding predicts improved outcomes. This information could be valuable in determining the best treatment strategy for different patients.

Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.

The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.

The Toxoplasma gondii Cyst Wall Protein CST1 Is Critical for Cyst Wall Integrity and Promotes Bradyzoite Persistence

Toxoplasma gondii infects up to one third of the world's population. A key to the success of T. gondii as a parasite is its ability to persist for the life of its host as bradyzoites within tissue cysts. The glycosylated cyst wall is the key structural feature that facilitates persistence and oral transmission of this parasite. Because most of the antibodies and reagents that recognize the cyst wall recognize carbohydrates, identification of the components of the cyst wall has been technically challenging. We have identified CST1 (TGME49_064660) as a 250 kDa SRS (SAG1 related sequence) domain protein with a large mucin-like domain. CST1 is responsible for the Dolichos biflorus Agglutinin (DBA) lectin binding characteristic of T. gondii cysts. Deletion of CST1 results in reduced cyst number and a fragile brain cyst phenotype characterized by a thinning and disruption of the underlying region of the cyst wall. These defects are reversed by complementation of CST1. Additional complementation experiments demonstrate that the CST1-mucin domain is necessary for the formation of a normal cyst wall structure, the ability of the cyst to resist mechanical stress, and binding of DBA to the cyst wall. RNA-seq transcriptome analysis demonstrated dysregulation of bradyzoite genes within the various cst1 mutants. These results indicate that CST1 functions as a key structural component that confers essential sturdiness to the T. gondii tissue cyst critical for persistence of bradyzoite forms.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and β-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcβ1-4)Galβ1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Altered glycosylation in peri-implantation phase endometrium in women with stages III and IV endometriosis

Endometriosis is a common cause of infertility and pelvic pain. Lectin histochemistry has shown that glycan expression is a sensitive marker of differentiation in the normal endometrium. Endometrial biopsies were taken during the implantation window from women with subfertility and advanced (stage III and IV) endometriosis to evaluate specific glycans bound by lectins from Dolichos biflorus agglutinin (DBA) and Vicia villosa agglutinin (VVA), which detect related but distinct glycan sequences regulated by progesterone action. Endometrial tissue from 12 women with subfertility and advanced endometriosis and 11 healthy controls were taken on days 19-24 of the menstrual cycle and processed into either epoxy resin or paraffin wax. Lectin histochemistry was analysed using light microscopy to quantify the amount of glandular reaction product. There was a significant (P = 0.011) reduction in DBA binding to endometrium from patients with endometriosis compared with controls, which was not seen with VVA (P = 0.135). Three stage IV biopsies and one stage III biopsy completely failed to bind DBA and, of these, three showed moderate glandular binding of VVA. DBA and VVA binding differed significantly (P= 0.0039) in the endometriosis specimens whereas in controls no significant difference was detected (P = 0.812). Secretory phase glycosylation in women with advanced endometriosis differs from that in healthy women with a reduction in fucosylated N-acetylgalactosamine sequences bound by DBA. Shorter VVA-binding glycans are not significantly affected. In addition to indicating abnormalities of epithelial differentiation, these findings may be directly relevant to implantation failure, as blastocyst attachment requires a critical interaction with the epithelial glycocalyx.

Case report: Diffuse splenic metastasis of occult breast cancer with incompatible blood group antigenic determinants

Cancer cells with immunogenic properties having altered protein glycosilation, modified blood group substances have been widely studied [Kannagi R, Miyake M, Zenita KM, Itai S, Hiraiwa N, Shigeta K, et al. Cancer-associated carbohydrate antigens: modified blood group substances and oncodevelopmental antigens on tumor cells. Gann Monogr Cancer Res 1988; 34: p. 15-28; Hakomori S. Antigen structure and genetic basis of histo-blood groups A, B and O their changes associated with human cancer. Biochem Biophys Acta 1999; 1473: p. 247-266; Brooks SA, Carter TM, Royle L, Harvey DJ, Fry SA, Kinch C, et al. Altered glycosilation of proteins in cancer: what is the potential for new anti-tumour strategies. Anticancer Agents Med Chem 2008; 8: p. 2-21]. In the study reported here, a 78-year-old female patient was admitted to the hospital with circulatory failure. At autopsy, the spleen (weight: 420 g) was extremely firm with a diffusely blackberry-colored cut surface. There were no signs of carcinomatous process at autopsy. By histology, the spleen showed diffuse metastatic carcinomatous infiltration. Using immunohistochemistry, an antibody to breast carcinoma antigen (BioGenex) labelled metastatic cells of the spleen and bone marrow. The patient was blood group O. Labelling for binding of lectins with and without blood group antigen specificity and monoclonal antibodies was carried out. The B blood group specific Banderiaea simplicifolia agglutinin I and an anti-B blood group monoclonal antibody labelled all the metastatic cells of spleen and bone marrow intensely. There was no detection of blood group A antigen by either binding of Dolichos biflorus agglutinin or anti-blood group A monoclonal antibodies. These observations raise the possibility that the detected incompatible B blood group antigen determinants on the metastatic cells were immunogenic. The surviving carcinoma cells may have found a place of refuge from immune surveillance in the spleen and in the bone marrow, where the complement-mediated tumor cell lysis immune rejection was not effective.

Lectin UEA-I-Binding Proteins Are Specifically Increased in the Squamous Epithelium of Patients with Barrett’s Esophagus

Background: Chronic gastritis and esophagitis are associated with changes in mucosal glycosylation patterns. Lectins represent a group of specific carbohydrate binding proteins that can be used as sensitive tools for the analysis of glycosylation patterns. Aim: To investigate the binding patterns of lectins Ulex europaeus agglutinin-I (UEA-I), Dolichos biflorus agglutinin (DBA), Helix pomatia agglutinin (HPA) and peanut agglutinin (PNA) at the gastroesophageal junction in nonerosive (NERD), erosive reflux disease (ERD) and Barrett’s esophagus (BE). Methods: One hundred and twenty-two patients (female n = 53; male n = 69; controls n = 28; NERD n = 36; ERD n = 24 and BE n = 34) were included in this study. The binding patterns of lectins were examined immunohistochemically at the squamocolumnar junction, in squamous epithelium and columnar-lined epithelium. Staining patterns of lectins were semiquantitatively evaluated using an immunohistochemical score and the data were analyzed using non-parametric tests. Results: BE, as compared to the controls, was associated with specific lectin-binding patterns: lectin binding of UEA-I and DBA were significantly decreased at the superficial (p = 0.012; p = 0.00036, respectively) and at the deep glandular body (p = 0.045; p = 0.055, respectively). Comparisons of lectin-staining scores between GERD and BE revealed significant increases of UEA-I in both the stratum superficiale (p = 0.0155) and stratum spinosum (p = 0.0048) of SE in patients with BE. Notable, this change was specific for patients with BE, while no difference was observed between patients with GERD and controls. Conclusion: We found two major types of lectin-binding patterns. First, lectin-binding characteristics associated with GERD in general, and second, lectin-binding patterns which were specific for BE. Lectin UEA-I-binding proteins were specifically increased in the squamous epithelium of patients with BE. Thus, UEA-I may serve as a potential marker for BE, especially in patients with short segment BE.

Peritoneal ectopic lesions from women with endometriosis show abnormalities in progesterone-dependent glycan expression

Examination of 12 paired peritoneal ectopic and eutopic endometria for histochemical binding of Dolichos biflorus agglutinin, normally found in the mid-late secretory part of the cycle, showed a failure of lectin binding in 9 of 11 secretory-phase lesions although the eutopic specimens generally stained normally. This failure of glycan expression in the secretory phase may result from various anomalies, including an inability to respond to progesterone, possibly due to a lack of, or to nonfunctional, progesterone receptors, suggesting that an ectopic environment may produce changes in tissue cell biology and hormonal responsiveness compared with that of eutopic endometrium.

Cultured Bovine Trophoblast Cells Differentially Express Genes Encoding Key Steroid Synthesis Enzymes

Placental trophoblasts are an important source of endocrine, paracrine and autocrine acting hormones. The aim of the present study was to establish and evaluate a tissue culture model for bovine trophoblasts to study regulation of key genes of steroid hormone synthesis. Trophoblast cells were isolated from cotyledons by collagenase disaggregation and subsequent percoll density gradient centrifugation. The cells were seeded on collagen coated dishes and incubated for up to seven days. The cells were characterized for the presence of mesenchymal vimentin and epithelial cytokeratin filaments and for Dolichos biflorus agglutinin (DBA) binding, a marker for differentiated trophoblast giant cells. Transcripts of Hsd3b, Cyp17 and Cyp19 encoding 3β-HSD, P450c17 and P450arom, the key enzymes of progesterone, androgen, and oestrogen biosynthesis, respectively, and of Csh1 encoding the trophoblast-specific hormone placental lactogen (PL) were measured by qPCR. Uninucleate cotyledonary epithelial cells and bi- and trinucleate trophoblast giant cells efficiently formed a dense cell layer on the collagen coated dishes within 24 h. Bi- and trinucleate cells showed DBA binding and weak or undetectable cytokeratin immunoreactivity. Vimentin-positive, fibroblast-like cells were found on top of this cell layer. Cyp19 transcripts were found in freshly dissociated but not in cultured cells. Cyp17 expression continuously increased, Hsd3b transcripts largely and rapidly increased during the first days in culture, followed by a decline after three days, whereas Csh1 decreased towards day seven. Serum free culture conditions significantly enhanced Cyp17 and Csh1 but not Hsd3b expression. The data indicate that collagen is a favourable substrate for cultured binucleate trophoblast giant cells. The cells represent an in vitro model to study the regulation of key genes of placental progesterone and androgen but not of oestrogen biosynthesis.

Identification, Isolation, and In Vitro Culture of Porcine Gonocytes1

Gonocytes are primitive germ cells that reside in the seminiferous tubules of neonatal testes and give rise to spermatogonia, thereby initiating spermatogenesis. Due to a lack of specific markers, the isolation and culture of these cells has proven to be difficult in the pig. In the present study, we show that a lectin, Dolichos biflorus agglutinin (DBA), which has specific affinity for primordial germ cells (PCGs) in the genital ridge, binds specifically to gonocytes in neonatal pig testes. The specific affinity of DBA for germ cells was progressively lost with age. This suggests that DBA binds strongly to primitive germ cells, such as gonocytes, weakly to primitive spermatogonia, and not at all to spermatogonia. The presence of alkaline phosphatase (AP) activity in the germ cells of neonatal pig testis confirmed the existence of primitive germ cells. Gonocytes from neonatal pig testis were purified, and a cell population that consisted of approximately 70% gonocytes was obtained, as indicated by the DBA binding assay. Purified gonocytes were cultured in DMEM/F12 supplemented with 10% FBS in the absence of any specific growth factors for 7 days. The cells remained viable and proliferated actively in culture. Initially, the gonocytes grew as focal colonies that transformed to three-dimensional colonies by 7 days of culture. Cultured germ cells expressed SSEA-1, a marker for embryonic stem (ES) cells, and were negative for the expression of somatic cell markers. These results should help to establish a male germ cell line that could be used for studying spermatogenesis in vitro and for genetic modification of pigs.

Primordial germ cell migration in the rat: preliminary evidence for a role of galactosyltransferase.

The precise cellular mechanism of primordial germ cell (PGC) migration remains unknown. Cell surface galactosyltransferase (GalTase) is known to play unique roles in the process of locomotion of many migratory cells. With an objective to seek evidence for possible involvement of GalTase in the migratory process of PGC, we evaluated germ cell migration in the rat following experimental modulation of embryonic GalTase activity. Pregnant rats were laparotomized under anesthesia on Day 10 of pregnancy. While embryos of one uterine horn received lysozyme (100 microg/fetus), those of the other received alpha-lactalbumin (LA; 100 microg/fetus), N-acetylglucosamine (GlcNAc; 250 nmole/fetus), uridine 5'-monophosphate (UMP; 2.5 micromole/fetus), uridine diphosphate-galactose (UDP-gal; 250 nmole/fetus), or a combination of 250 nmole of UDP-gal and 2.5 micromole of UMP/fetus. Between gestation Days 12 and 14, embryos were dissected out and processed for histochemical localization of PGC on the basis of binding of Dolichos biflorus agglutinin on the surface glycoconjugate of the germ cells. The number of PGC in each embryo was counted. There was a daywise increase in the number of PGC in all groups. As compared with lysozyme-exposed controls, the numbers of PGCs at the day-specific sites on all days of examination were significantly lower in the LA- as well as GlcNAc-exposed groups. UMP or UDP-gal individually exerted little or no influence, while the total PGC count rose significantly over the respective control values under simultaneous exposure to UMP and UDP-gal. The present findings suggest a likely catalytic role of GalTase in the process of germ cell migration.

Relationships between degree of binding, cytotoxicity and cytoagglutinating activity of plant-derived agglutinins in normal lymphocytes and cultured leukemic cell lines.

To clarify the relationships between the degree of lectin-cell binding, cytotoxicity and cytoagglutinating activity of plant-derived lectins in normal lymphocytes and cultured leukemic cell lines. Plant lectins with different quaternary structures and saccharide specificity were used: Dolichos biflorus agglutinin (DBA), Soybean agglutinin (SBA) and Wheat germ agglutinin (WGA). The leukemic cell lines used were: Jurkat, MOLT-4, RPMI-8402, HPB-ALL, CCR-HSB-2 and BALL-1 (derived from acute lymphoblastic leukemia); Raji and Daudi (derived from Burkitt's lymphoma); K-562 (derived from myelogenous leukemia). The lectin-cell binding was detected microscopically and fluorimetrically using FITC-conjugated lectins. Cytotoxicity was estimated by the CellTiter-Glo luminescent cell viability assay, and cytoagglutinating activity by a spectrophotometric method. The binding of DBA and SBA to normal lymphocytes was negligible, while their binding to leukemic cells increased markedly with increasing lectin concentration. Analogous results were obtained for WGA. However, it was found that WGA also interacted to a significant degree with normal lymphocytes. The degree of lectin-cell binding increased in the order: DBA<SBA<WGA. The cytoagglutinating activity and cytotoxicity of lectins increased in the same order. DBA did not exhibit a cytotoxic effect against normal or leukemic cells, and showed a poor cytoagglutinating activity only in MOLT-4, CCR-HSB-2 and BALL-1 cells. SBA exhibited poor cytotoxicity against Jurkat, RPMI-8402, HPB-ALL and CCR-HSB-2 cells, but a well-defined cytotoxicity against Raji and Daudi cells. SBA showed poor cytoagglutinating activity in leukemic cells. In contrast, WGA at concentrations higher than 0.05 microM showed high cytotoxicity against all leukemic cell lines tested as well as against normal lymphocytes. WGA also showed a well-expressed cytoagglutinating effect in all cell lines except normal lymphocytes. There was a moderate inverse correlation between cell viability and the velocity of cytoagglutination ( r=-0.56, P<0.001), and a good correlation between cell viability and the degree of lectin-cell binding ( r=-0.75, P<0.001). There was a low positive correlation between the velocity of cytoagglutination and the degree of lectin-cell binding ( r=0.43, P<0.001). The results suggest that the lectins that bound most strongly to leukemic cells expressed higher cytotoxic and cytoagglutinating activities.

Prenatal exposure to high galactose adversely affects initial gonadal pool of germ cells in rats.

In rats, prenatal exposure to high concentrations of galactose may contribute to a condition that is equivalent to the premature ovarian failure (POF) component of human galactosaemia. We investigated if development of POF under experimental galactosaemia-like conditions was attributed to impaired germ cell migration. Pregnant rats were fed pellets supplemented with, or without, 35% galactose from day 3 of conception continuing through parturition. Between days 12-15, embryos from one uterine horn were dissected out. Primordial germ cells (PGC) were histochemically localized and counted on the basis of binding of Dolichos biflorus agglutinin, a lectin specific for terminal N-acetylgalactosamine (GalNAc), to the surface glycoconjugate of the germ cells. The embryos from the other uterine horn were maintained until parturition. Liver activity of uridine diphosphate galactose 4-epimerase, the enzyme involved at multiple steps in the process of synthesis of GalNAc, was assayed in 1-2 day old female pups. The numbers of PGC at the day-specific sites on all days of examination were significantly lower (P </= 0.0003), and liver epimerase activity was significantly (P = 0.000001) reduced in the galactose-exposed group. Impaired germ cell migration leading to the development of gonads with deficient initial pools of germ cells may form the causal link between galactosaemia and POF.

Expression of CD105 (endoglin) in arteriolar endothelial cells of human endometrium throughout the menstrual cycle.

The cellular mechanisms underlying normal and pathological endometrial bleeding are not well understood, although abnormalities in the structure of endometrial blood vessels may lead to menstrual disorders. Endothelial cells in different organs are heterogeneous and differ in structure, function, antigen composition, metabolic properties and responses to growth factors. Immunostaining was performed with anti-CD105, CD31, CD34 and von Willebrand factor (vWF), and lectin binding with Ulex europaeus agglutinin 1 (UEA 1), Bandeieraea simplicifolia agglutinin 1 (BS 1), Dolichos biflorus agglutinin (DBA) and Peanut agglutinin (PNA) to characterize endothelial cells in human endometrium throughout the menstrual cycle. Serial sections fixed with formalin were stained with primary antibodies and lectins after antigen retrieval. Positive staining for CD31, CD105 and vWF was confined to the vascular endothelium. Endothelial expression of CD31 was observed in all types of vessel, including single cells, and strong staining was found during the early proliferative and mid-secretory phases. Anti-vWF stained arterioles and veins, but there was little positive staining of capillaries. In contrast, staining for CD105 was confined to the arterioles. Although anti-CD34 strongly stained endothelial cells of small vessels and capillaries, staining was also observed on some non-endothelial stromal cells. Strong positive staining for UEA 1 was observed in endothelial cells of all types of vessel throughout the menstrual cycle. Binding of PNA, DBA and BS 1 was confined to the apical region of glandular epithelial cells. This study demonstrates that the differential binding of anti-CD31, CD34, CD105, vWF and UEA 1 distinguishes between endometrial populations of endothelial cells.

Association of Dolichos biflorus lectin binding with full differentiation of bovine trophoblast cells

Binucleate cells in ruminant placenta are differentiated from fetal mononucleate trophoblast cells and secrete many glycoproteins. This study characterized bovine placental binucleate cells in primary culture and a bovine trophoblastic cell line (BT-1) with an N-acetyl-galactosamine-binding lectin, Dolichos biflorus agglutinin (DBA). DBA specifically bound to the surface membrane and the cytoplasm of binucleate cells. Mononucleate epithelial cells and fibroblasts were free of DBA. DBA-positive binucleate cells corresponded to the fully matured cells, producing placental lactogen, and the cytoplasm was devoid of cytokeratin. Binucleate cells assumed a flattened shape on a collagen substratum in an extended culture, and entered a dedifferentiated state with a degranulation of placental lactogen. In these flattened cells, DBA reactions were attenuated in the cytoplasm. DBA binding in BT-1 was subsequently examined. BT-1 was derived from blastocysts produced in vitro and is trophectodermal as shown by the expression of cytokeratin. BT-1 was able to differentiate into placental lactogen-producing binucleate cells on a collagen gel substratum. Cytokeratin expression in BT-1 was downregulated with the differentiation into binucleate cells. However, DBA bound to neither mononucleate nor the differentiated binucleate cells in BT-1. These results indicate that binucleate cells in vivo but not binucleate cells derived from BT-1, specifically developed glycoconjugates recognized by DBA. The glycoconjugate expression was associated with fully differentiated cells. The onset of DBA binding in binucleate cells coincides with placental development, and binucleate cells differentiated from BT-1 cell cultures may reflect those cells at earlier stages of gestation.

Association of Dolichos biflorus lectin binding with full differentiation of bovine trophoblast cells

Binucleate cells in ruminant placenta are differentiated from fetal mononucleate trophoblast cells and secrete many glycoproteins. This study characterized bovine placental binucleate cells in primary culture and a bovine trophoblastic cell line (BT-1) with an N-acetyl-galactosamine-binding lectin, Dolichos biflorus agglutinin (DBA). DBA specifically bound to the surface membrane and the cytoplasm of binucleate cells. Mononucleate epithelial cells and fibroblasts were free of DBA. DBA-positive binucleate cells corresponded to the fully matured cells, producing placental lactogen, and the cytoplasm was devoid of cytokeratin. Binucleate cells assumed a flattened shape on a collagen substratum in an extended culture, and entered a dedifferentiated state with a degranulation of placental lactogen. In these flattened cells, DBA reactions were attenuated in the cytoplasm. DBA binding in BT-1 was subsequently examined. BT-1 was derived from blastocysts produced in vitro and is trophectodermal as shown by the expression of cytokeratin. BT-1 was able to differentiate into placental lactogen-producing binucleate cells on a collagen gel substratum. Cytokeratin expression in BT-1 was downregulated with the differentiation into binucleate cells. However, DBA bound to neither mononucleate nor the differentiated binucleate cells in BT-1. These results indicate that binucleate cells in vivo but not binucleate cells derived from BT-1, specifically developed glycoconjugates recognized by DBA. The glycoconjugate expression was associated with fully differentiated cells. The onset of DBA binding in binucleate cells coincides with placental development, and binucleate cells differentiated from BT-1 cell cultures may reflect those cells at earlier stages of gestation.

Postmitotic basal cells in squamous cell epithelia are identified with Dolichos biflorus agglutinin – functional consequencesNote

Dolichos biflorus agglutinin (DBA) is a plant lectin specifically recognizing alpha-N-acetylgalactosamine. Controversial reports regarding the binding of DBA to the epidermis have been published. Using a double labeling procedure at the single-cell level, we studied the expression of DBA-reactive binding sites in conjunction with markers of cell proliferation and differentiation in normal human epidermis, cornea, and malignant tumors as well as in cultured keratinocytes. The results characterize the cells recognized by DBA as postmitotic early differentiating cells, identifiable by their lack of expression of the proliferation marker (Ki-67). The Golgi complex of a limited number of cultured keratinocytes was recognized by DBA and some of these cells show the accumulation of beta1 integrin chain in the Golgi complex. This process seems to be important for the migration of postmitotic cells from the basal to the suprabasal layers.