Bandeiraea Simplicifolia Lectin Research Articles

Regulation of Parietal Cell Homeostasis by Bone Morphogenetic Protein Signaling

<h3>Background and Aims</h3> Loss of bone morphogenetic protein (BMP) signaling in the stomach, achieved by transgenic expression of the BMP inhibitor noggin (<i>H</i>⁺<i>/K</i>⁺<i>-Nog</i> mice), causes parietal cell (PC) loss, spasmolytic polypeptide-expressing metaplasia, a marker of preneoplasia, and activation of cell proliferation. We examined if specific inhibition of BMP signaling in PCs leads to aberrations in epithelial homeostasis. <h3>Methods</h3> Mice with floxed alleles of BMP receptor 1a (<i>Bmpr1a</i>^flox/flox mice) were crossed to <i>H</i>⁺<i>/K</i>⁺<i>-Cre</i> mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Cre;Bmpr1a</i>^flox/flox mice. Morphology of the mucosa was analyzed by hematoxylin and eosin staining. Distribution of H⁺/K⁺-ATPase-, IF-, and Ki-67-positive cells was analyzed by immunostaining. Expression of pit and neck cell mucins was determined by staining with the lectins Ulex Europaeus Agglutinin 1 and Griffonia (Bandeiraea) simplicifolia lectin II, respectively. Isolation of PCs from control and <i>Nog</i>-expressing mice was achieved by crossing <i>H</i>⁺<i>/K</i>⁺<i>-Nog</i> mice to <i>Rosa26-tdTomato</i> (Tom) mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice. <i>H</i>⁺<i>/K</i>⁺<i>-Cre</i> mice were then crossed to <i>H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice to generate <i>H</i>⁺<i>/K</i>⁺<i>-Cre;H</i>⁺<i>/K</i>⁺<i>-Nog;Rosa26-tdTom</i> mice. Tom-labeled PCs were purified by flow cytometry. Changes in PC transcripts were measured by RNA-Seq. <h3>Results</h3> Six-month-old <i>H</i>⁺<i>/K</i>⁺<i>-Cre;Bmpr1a</i>^flox/flox mice exhibited increased epithelial cell proliferation, presence of transitional cells showing colocalization of IF with both Griffonia (Bandeiraea) simplicifolia lectin II-binding mucins and the H⁺/K⁺-ATPase, and expansion of Ulex Europaeus Agglutinin 1-positive cells. PC transcripts from Nog-expressing mice demonstrated induction of markers of Spasmolytic Polypeptide-Expressing Metaplasia. <h3>Conclusion</h3> PC-specific loss of BMP signaling alters the homeostasis of the gastric epithelium leading to the development of metaplasia.

Integrated glycomics strategy for galactosylated-N-glycans recognized by Bandeiraea Simplicifolia Lectin I in salivary proteins associated with lung cancer.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide, mainly due to late diagnosis and poor prognosis. Saliva is an important source for discovering biomarkers and contains an abundance of biological information. The purpose of this study was to determine whether galactosylation levels of salivary proteins are associated with LC. First, we analyzed the alterations of the glycopatterns recognized by Bandeiraea Simplicifolia Lectin I (BS-I) in five groups (healthy volunteers [HV]: 28, benign pulmonary disease [BPD]: 27, lung adenocarcinoma [ADC]: 39, squamous cell carcinoma [SCC]: 28, small-cell lung cancer [SCLC]: 22) of 144 saliva samples using lectin microarrays. Pooled samples from each group were subsequently validated by the lectin blotting technique. Finally, the N-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from pooled samples for each group were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with LC compared with BPD and HV. Receiver operating characteristic (ROC) analysis indicated that the levels of salivary glycopattern recognized by BS-I could discriminate lung disease (BPD, ADC, SCC, and SCLC) and HV with an AUC of 0.700 (95% CI: 0.589-0.812), and discriminate LC and BPD with an AUC of 0.860 (95% CI: 0.763-0.956). Also, the proportion of galactosylated N-glycans in ADC (38.4%), SCC (43.1%), and SCLC (39.5%) increased compared to HV (30.1%) and BPD (33.7%), and two galactosylated N-glycan peaks (m/z 1828.683, 2418.853) could be identified only in the LC groups (ADC, SCC, and SCLC). These findings could provide crucial information on galactosylated N-linked glycans associated with LC and facilitate the study of LC biomarkers based on precise alterations of galactosylated N-glycans in saliva.

Poly (ethylene) glycol (PEG) precipitation of glycosylated and non-glycosylated monoclonal antibodies

The solubility of monoclonal antibodies (mAb) affects their production and their intravenous administration to patients. In this work, the solubility of a fully glycosylated and a non-glycosylated human mAb expressed in corn was studied by inducing their precipitation by poly(ethylene) glycol (PEG). The experiments were done using PEG 1450 and 8000 at concentrations ranging from 0% to 30% w/w, at different pHs and temperatures. Additional studies were performed in the presence of Griffonia (Bandeiraea) simplicifolia Lectin II, which binds to glycosylated proteins. These studies show that glycosylation increases the solubility of the antibody and that models based on excluded volume principles or on the statistical correlation of solubilities are unable to capture the effect of glycosylation on protein precipitation by PEG. PEG molecular weight controls the onset point of the precipitation curves but glycosylation is the main effect on PEG precipitation efficiency. A two-stages precipitation pattern was observed when a lectin and PEG were added to the glycosylated or non-glycosylated mAbs.

Abnormal Galactosylated-Glycans recognized by Bandeiraea Simplicifolia Lectin I in saliva of patients with breast Cancer.

Currently, the definitive diagnosis in breast cancer requires biopsy and histopathology, such the most effective markers are tissue-based. However, the advantages of saliva in collection and storage make it possible for assessing human pathology and contributing to the development of cancer-related biomarkers for clinical application. The present study validated alteration of salivary protein glycopatterns recognized by Bandeiraea simplicifolia lectin I (BS-I) in the saliva of patients with breast diseases using saliva microarrays, and the N/O-glycan profiles of their salivary glycoproteins isolated by the BS-I-magnetic particle conjugates from 259 female subjects (66 healthy volunteers (HV), 65 benign breast cyst or tumor patients (BB), 66 patients with breast cancer in stage I (BC-I) and 62 patients with breast cancer in stage II (BC-II)) were analyzed by MALDI-TOF/TOF-MS. The results showed that the expression level of galactosylated glycans recognized by BS-I was significantly increased in patients with breast cancer compared with HV (p < 0.05). Totally, there were 11/10, 10/19, 7/24 and 7/9 galactosylated N-/O-linked glycans were identified and annotated from the pooled salivary samples of HV, BB, BC-I and BC-II, respectively. One galactosylated N-glycan peak (m/z 2773.977), and 4 galactosylated O-glycan peaks (m/z 868.295, 882.243, 884.270 and 1030.348) were found only in BC-I. These findings could provide pivotal information on galactosylated N/O-linked glycans related to breast cancer, and promote the study of biomarkers for early-stage breast cancer based on precise alterations of galactosylated N/O-glycans in saliva.

Glycan changes in the olfactory mucosa of rats with experimental autoimmune encephalomyelitis

Glycans are components of glycoconjugates and function in odorant recognition and cell signaling in the olfactory mucosa. However, little is known about glycan expression in the olfactory mucosa in the presence of neuroinflammatory disorders, which can influence olfaction. We evaluated the changes in glycan in the olfactory mucosa of rats with experimental autoimmune encephalomyelitis (EAE) by histochemical analyses of 21 lectins. In the olfactory mucosa of normal control rats, 16 lectins bound to olfactory sensory neurons, supporting cells, basal cells, nerve and Bowman’s glands, and their expression did not significantly change during the course of EAE. In rats with paralytic-stage EAE, five lectins showed different reactivities with the olfactory mucosa compared to those of normal control rats. Of them, Bandeiraea simplicifolia lectin (BSL)-II and BSL-I showed transiently downregulated binding to olfactory sensory neurons and supporting cells in rats with EAE. The reactivities of Lens culinaris agglutinin for the basement membrane, Vicia villosa agglutinin for Bowman’s glands and Dolichos biflorus agglutinin for all nuclei were upregulated in the olfactory mucosa of EAE rats. These results suggest that BSL-II-binding N-acetyl-glucosamine and BSL-I-binding N-acetyl-galactose are involved in transient olfactory dysfunction in EAE, which may hamper odor perception and/or signal processing in olfactory sensory neurons.

Muc5ac null mice are predisposed to spontaneous gastric antro-pyloric hyperplasia and adenomas coupled with attenuated H.pylori-induced corpus mucous metaplasia

Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide and is strongly associated with chronic Helicobacter pylori (Hp) infection. The ability of Hp to closely adhere to the gastric surface protective mucous layer containing mucins (MUC in humans and Muc in animals), primarily Muc5ac, is integral in the stepwise pathogenesis from gastritis to cancer. To probe the role of Muc5ac in Hp-induced gastric pathology, Muc5ac−/− and Muc5ac+/+ (WT) mice were experimentally infected with Hp Sydney strain (SS1). At 16 weeks and 32 weeks post infection (wpi), groups of mice were euthanized and evaluated for the following: gastric histopathological parameters, immunohistochemical expression of mucins (Muc5ac, Muc1, Muc2), Trefoil factor family proteins (Tff1 and Tff2), Griffonia (Bandeiraea) simplicifolia lectin II (GSL II) (mucous metaplasia marker) and Clusterin (Spasmolytic Polypeptide Expressing Metaplasia (SPEM) marker), Hp colonization density by qPCR and gastric cytokine mRNA levels. Our results demonstrate that Muc5ac−/− mice developed spontaneous antro-pyloric proliferation, adenomas and in one case with neuroendocrine differentiation; these findings were independent of Hp infection along with strong expression levels of Tff1, Tff2 and Muc1. Hp-infected Muc5ac−/− mice had significantly lowered gastric corpus mucous metaplasia at 16 wpi and 32 wpi (P = 0.0057 and P = 0.0016, respectively), with a slight reduction in overall gastric corpus pathology. GSII-positive mucous neck cells were decreased in Hp-infected Muc5ac−/− mice compared to WT mice and clusterin positivity was noted within metaplastic glands in both genotypes following Hp infection. Additionally, Hp colonization densities were significantly higher in Muc5ac−/− mice compared to WT at 16 wpi in both sexes (P = 0.05) along with a significant reduction in gastric Tnfα (16 wpi—males and females, P = 0.017 and P = 0.036, respectively and 32 wpi—males only, P = 0.025) and Il-17a (16 wpi—males) (P = 0.025). Taken together, our findings suggest a protective role for MUC5AC/Muc5ac in maintaining gastric antral equilibrium and inhibiting Hp colonization and associated inflammatory pathology.

Genetic Insufficiency of 12/15LOX in Mice‐Facilitated Angiogenic Response Post‐Myocardial Infarction

12/15 lipoxygenase (12/15LOX) is a fat‐metabolizing enzyme that uses different fatty acids to form bioactive lipids in pathology of diseases such as myocardial infarction (MI)‐induced heart failure (HF). 12/15LOX‐null mice show an effective healing pattern with a higher survival rate as compared to wildtype (WT), but the mechanism is unknown. We aimed to test whether 12/15LOX expression in wildtype mice impairs angiogenesis of infarcted myocardium post‐MI in acute and chronic HF. MI was surgically induced in wildtype (WT; C57BL/6) and 12/15LOX‐null mice (2–4 months old) by ligating the left anterior descending artery. Mice LV tissues were stained using biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin at acute (day; d1 and d5) and chronic (d28 and d56) time points suggestive of chronic HF post‐MI. To determine angiogenesis, LV 40X images of the remote and peri‐infarct regions were analyzed using light microscopy and then quantified blood vessel density (BVD). As expected, non‐MI naive controls (d0) had the largest BVD compared to remote and peri‐infarct regions of WT and 12/15LOX‐deficient mice post‐MI. BVD diminished in WT and 12/15LOX‐deficient mice at d1. In the remote region, 12/15LOX‐deficient mice had a higher BVD than WT on d5, d28 and d56. BVD peaked on d5 for 12/15LOX‐deficient mice and on d28 for WT. In the peri‐infarct region, 12/15LOX‐deficient mice showed higher BVD than WT on days 1, 5, 28 and 56. BVD peaked on d5 in 12/15LOX‐deficient and WT mice post‐MI. BVD data indicated that angiogenesis peaks on day 5 (during the acute phase) in 12/15LOX‐deficient mice as compared to WT post‐MI. In summary, the presented results suggest that 12/15LOX delay angiogenesis post‐MI in HF.Support or Funding InformationThis research was supported in part by the American Physiology Society to MC as a STRIDE fellow and a grant from the National Heart, Lung and Blood Institute to GVH (HL132989).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Regulation of Gastric Lgr5+ve Cell Homeostasis by Bone Morphogenetic Protein (BMP) Signaling and Inflammatory Stimuli.

Background & AimsGastric Leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) cells exert important functions during injury and homeostasis. Bone morphogenetic protein (BMP) signaling regulates gastric inflammation and epithelial homeostasis. We investigated if BMP signaling controls the fate of Lgr5+ve cells during inflammation.MethodsThe H+/K+-adenosine triphosphatase β-subunit promoter was used to express the BMP inhibitor noggin (Nog) in the stomach (H+/K+-Nog mice). Inhibition of BMP signaling in Lgr5 cells was achieved by crossing Lgr5-EGFP-ires-CreERT2 (Lgr5-Cre) mice to mice with floxed alleles of BMP receptor 1A (Lgr5-Cre;Bmpr1aflox/flox mice). Lgr5/GFP+ve cells were isolated using flow cytometry. Lineage tracing studies were conducted by crossing Lgr5-Cre mice to mice that express Nog and tdTomato (Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom). Infection with Helicobacter felis was used to induce inflammation. Morphology of the mucosa was analyzed by H&E staining. Distribution of H+/K+-adenosine triphosphatase-, IF-, Ki67-, CD44-, CD44v9-, and bromodeoxyuridine-positive cells was analyzed by immunostaining. Expression of neck and pit cell mucins was determined by staining with the lectins Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, respectively. Id1, Bmpr1a, Lgr5, c-Myc, and Cd44 messenger RNAs were measured by quantitative reverse-transcription polymerase chain reaction.ResultsLgr5-Cre;Bmpr1aflox/flox mice showed diminished expression of Bmpr1a in Lgr5/GFP+ve cells. Infection of Lgr5-Cre;Bmpr1aflox/flox mice with H felis led to enhanced inflammation, increased cell proliferation, parietal cell loss, and to the development of metaplasia and dysplasia. Infected Lgr5-Cre;H+/K+-Nog;Rosa26-tdTom mice, but not control mice, showed the presence of tomato+ve glands lining the lesser curvature that stained positively with Griffonia (Bandeiraea) simplicifolia lectin II and Ulex europaeus agglutinin 1, and with anti-IF, -CD44, -CD44v9, and -bromodeoxyuridine antibodies.ConclusionsInflammation and inhibition of BMP signaling activate Lgr5+ve cells, which give rise to metaplastic, dysplastic, proliferating lineages that express markers of mucus neck and zymogenic cell differentiation.

Macrophage overexpression of matrix metalloproteinase-9 in aged mice improves diastolic physiology and cardiac wound healing after myocardial infarction.

Matrix metalloproteinase (MMP)-9 increases in the myocardium with advanced age and after myocardial infarction (MI). Because young transgenic (TG) mice overexpressing human MMP-9 only in macrophages show better outcomes post-MI, whereas aged TG mice show a worse aging phenotype, we wanted to evaluate the effect of aging superimposed on MI to see if the detrimental effect of aging counteracted the benefits of macrophage MMP-9 overexpression. We used 17- to 28-mo-old male and female C57BL/6J wild-type (WT) and TG mice ( n = 10-21 mice/group) to evaluate the effects of aging superimposed on MI. Despite similar infarct areas and mortality rates at day 7 post-MI, aging TG mice showed improved diastolic properties and remodeling index compared with WT mice (both P < 0.05). Macrophage numbers were higher in TG than WT mice at days 0 and 7 post-MI, and the post-MI increase was due to elevated cluster of differentiation 18 protein levels (all P < 0.05). RNA sequencing analysis of cardiac macrophages isolated from day 7 post-MI infarcts identified 1,276 statistically different (all P < 0.05) genes (994 increased and 282 decreased in TG mice). Reduced expression of vascular endothelial growth factor A, platelet-derived growth factor subunit A, and transforming growth factor-β3, along with elevated expression of tissue inhibitor of MMP-4, in macrophages revealed mechanisms of indirect downstream effects on fibroblasts and neovascularization. While collagen accumulation was enhanced in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both), the post-MI collagen cross-linking ratio was higher in WT mice ( P < 0.05), consistent with increased diastolic volumes. Vessel numbers [by Griffonia ( Bandeiraea) simplicifolia lectin I staining] were decreased in TG mice compared with WT mice at days 0 and 7 post-MI ( P < 0.05 for both). In conclusion, macrophage-derived MMP-9 improved post-MI cardiac wound healing through direct and indirect mechanisms to improve diastolic physiology and remodeling. NEW & NOTEWORTHY Aging mice with macrophage overexpression of matrix metalloproteinase-9 have increased macrophage numbers 7 days after myocardial infarction, resulting in improved diastolic physiology and left ventricular remodeling through effects on cardiac wound healing.

Differential Lectin Binding Patterns Identify Distinct Heart Regions in Giant Danio ( Devario aequipinnatus) and Zebrafish ( Danio rerio) Hearts.

Lectins are carbohydrate-binding proteins commonly used as biochemical and histochemical tools to study glycoconjugate (glycoproteins, glycolipids) expression patterns in cells, tissues, including mammalian hearts. However, lectins have received little attention in zebrafish ( Danio rerio) and giant danio ( Devario aequipinnatus) heart studies. Here, we sought to determine the binding patterns of six commonly used lectins-wheat germ agglutinin (WGA), Ulex europaeus agglutinin, Bandeiraea simplicifolia lectin (BS lectin), concanavalin A (Con A), Ricinus communis agglutinin I (RCA I), and Lycopersicon esculentum agglutinin (tomato lectin)-in these hearts. Con A showed broad staining in the myocardium. WGA stained cardiac myocyte borders, with binding markedly stronger in the compact heart and bulbus. BS lectin, which stained giant danio coronaries, was used to measure vascular reconstruction during regeneration. However, BS lectin reacted poorly in zebrafish. RCA I stained the compact heart of both fish. Tomato lectin stained the giant danio, and while low reactivity was seen in the zebrafish ventricle, staining was observed in their transitional cardiac myocytes. In addition, we observed unique staining patterns in the developing zebrafish heart. Lectins' ability to reveal differential glycoconjugate expression in giant danio and zebrafish hearts suggests they can serve as simple but important tools in studies of developing, adult, and regenerating fish hearts.

Corpus luteal angiogenesis in a high milk production dairy breed differs from that of cattle with lower milk production levels

Globally, high producing, young dairy cows often have health and fertility problems such as ovarian, uterine and placental dysfunction, mastitis and impaired wound healing. These health issues can result in a shortened average lifespan of fewer than three lactations. We hypothesise that many of these health issues may be an effect of altered angiogenesis. Hence, the aim of this pilot study was to investigate the status of vascularisation in the corpus luteum of a high milk-producing cattle breed (Holstein Friesian) compared with that of low milk producing beef cattle (Limousin) and of dual purpose cattle (Fleckvieh, Rotbunt). The corpus luteum was chosen because as a transient endocrine gland it is one of the few tissues with physiological angiogenesis in the adult. Blood vessels were labelled in paraffin sections of corpora lutea with the lectin Bandeiraea simplicifolia agglutinin I and the following angiogenesis parameters were analysed morphometrically using image analysis: (a) number of blood capillaries per mm&lt;sup&gt;2&lt;/sup&gt;, (b) intercapillary distance, (c) percentage area occupied by blood vessels and (d) area of blood vessel lumina (μm&lt;sup&gt;2&lt;/sup&gt;). This analysis revealed that the extent of corpus luteal vascularisation is greater in high milk-producing cattle than in beef and dual purpose cattle as shown by a significantly higher number of blood capillaries per mm², significantly shorter intercapillary distances and a higher percentage of the corpus luteum area covered by blood vessels (non-significant). Only the average value of the luminal area of the luteal blood capillaries in the high milk-producing cattle is smaller than that measured in the low producing cattle.

Differential Lectin Binding and Coronary Angiography in a Danio and non‐Model Fish Species

Lectins are carbohydrate‐binding proteins commonly used in the study of mammalian cells and tissues. However lectins have received little use in fish models and non‐models species. Here, we determine the binding patterns of commonly used lectins, including wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA lectin), Bandeiraea simplicifolia lectin (BS lectin), Concanavalin A (Con A), Ricinus communis (RCA), and Lycopersicon esculentum lectin (tomato lectin) in zebrafish and giant danio hearts. Our results show that WGA stained fish cardiac myocyte borders in patterns similar to that seen in mammalian hearts, with staining markedly stronger in the fish compact hearts. Con A showed broad and strong staining in fish ventricles. Interestingly, BS lectin reacted poorly to a number of fish species including zebrafish, pearl danio, common koi, goldfish, and Buenos Aires tetra. By contrast BS lectin strongly stained coronary vessels of giant danio, and several pleco and gourami species. Importantly, enhanced BS lectin binding to vascular endothelium compared to endocardium allowed for 3D coronary angiography in the giant danio by confocal and lightsheet microscopy, and for quantitation of vascular reconstruction during regeneration. In conclusion, our study demonstrates that lectins are simple but important tools for studies in models and non‐models fish species, and in adult and regenerating giant danio heart.Support or Funding InformationR15 HD084262‐01, FDC‐PDF‐DePauw University

Differential Lectin Binding in Giant Danio, Goldfish, and Zebrafish hearts

Lectins are carbohydrate‐binding proteins commonly used in the study of mammalian cells and tissues. However lectins have received little use in fish models. Here, we determine the binding patterns of three commonly used lectins: wheat germ agglutinin (WGA), Ulex europaeus agglutinin (UEA lectin), Bandeiraea simplicifolia lectin (BS lectin), in giant danio (GD), zebrafish (ZF), and goldfish (GF) hearts. Our results show that WGA stained cardiac myocyte borders of all three fish in patterns similar to that seen in mammalian hearts, with staining markedly stronger in the fish compact hearts. By contrast, UEA lectin, an endothelial cell marker in mammalian vascular beds, showed little to no binding to the coronary vasculature of these fish. Interestingly, BS lectin reacted poorly to the zebrafish coronary vasculature similar to our previously reported study in the goldfish. However BS lectin strongly stained coronary vessels of the Giant danio. Importantly, BS lectin biding was particularly strong in the vascular endothelium compared to endocardium of the Giant danio. This differential regional staining allowed for coronary angiography of the Giant danio heart and 3D reconstruction by confocal and light sheet fluorescence microscopy. In addition we were able to quantify coronary revascularization following cautery injury and during repair and regeneration of the Giant danio heart. Our results demonstrate that lectins differentially bind to coronary vasculature of fish and can be used as simple but powerful tools for the study of adult and regenerating fish hearts.

Vascular perfusion with fluorescent labeled lectin to study ovarian functions

The aim of this study was to optimize a method to visualize tissue vascularity by perfusing the local vascular bed with a fluorescently labeled lectin, combined with immunofluorescent labeling of selected vascular/tissue markers. Ovaries with the pedicle were obtained from adult non-pregnant ewes. Immediately after collection, the ovarian artery was perfused with phosphate buffered saline (PBS) to remove blood cells, followed by perfusion with PBS containing fluorescently labeled Griffonia (Bandeiraea) simplicifolia (BS1) lectin. Then, half of ovary was fixed in formalin and another half in Carnoy's fixative. BS1 was detected in blood vessels in ovaries fixed in formalin, but not in Carnoy's fixative. Formalin fixed tissue was used for immunofluorescence staining of two markers of tissue function and/or structure, Ki67 and smooth muscle cell actin (SMCA). Ki67 was detected in granulosa and theca cells, luteal and stromal tissue, and a portion of Ki67 staining was co-localized with blood vessels. SMCA was detected in pericytes within the capillary system, in blood vessels in all ovarian compartments, and in the stroma. Thus, blood vessel perfusion with fluorescently labeled lectin combined with immunohistochemistry, microscopy, and imaging techniques provide an excellent tool to study angiogenesis, vascular architecture, and organ structures and function in physiological and pathological conditions.

Studies on conjugation of Spirogyra using monoclonal culture

We succeeded in inducing conjugation of Spirogyra castanacea by incubating algal filaments on agar plate. Conjugation could be induced using clone culture. The scalariform conjugation was generally observed, while lateral conjugation was rarely. When two filaments formed scalariform conjugation, all cells of one filament behaved as male and those of other filament did as female. Very rarely, however, zygospores were formed in both of pair filaments. The surface of conjugation tube was stained with fluorescently labeled-lectins, such as Bandeiraea (Griffonia) simplicifolia lectin (BSL-I) and jacalin. BSL-I strongly stained the conjugation tubes, while weakly did the cell surface of female gamete first and then that of male gamete. Jacalin stained mainly the conjugation tubes. Addition of jacalin inhibited the formation of papilla, suggesting some important role of jacalin-binding material at the initial step of formation of the conjugation tubes.

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects. Previous studies have described techniques used to label the neonatal and adult thymus vasculature. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1-isolectin B₄) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-β to label thymic perivascular mesenchyme. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart.

A Major Fraction of Glycosphingolipids in Model and Cellular Cholesterol-containing Membranes Is Undetectable by Their Binding Proteins

Glycosphingolipids (GSLs) accumulate in cholesterol-enriched cell membrane domains and provide receptors for protein ligands. Lipid-based "aglycone" interactions can influence GSL carbohydrate epitope presentation. To evaluate this relationship, Verotoxin binding its receptor GSL, globotriaosyl ceramide (Gb(3)), was analyzed in simple GSL/cholesterol, detergent-resistant membrane vesicles by equilibrium density gradient centrifugation. Vesicles separated into two Gb(3/)cholesterol-containing populations. The lighter, minor fraction (<5% total GSL), bound VT1, VT2, IgG/IgM mAb anti-Gb(3), HIVgp120 or Bandeiraea simplicifolia lectin. Only IgM anti-Gb(3), more tolerant of carbohydrate modification, bound both vesicle fractions. Post-embedding cryo-immuno-EM confirmed these results. This appears to be a general GSL-cholesterol property, because similar receptor-inactive vesicles were separated for other GSL-protein ligand systems; cholera toxin (CTx)-GM1, HIVgp120-galactosyl ceramide/sulfatide. Inclusion of galactosyl or glucosyl ceramide (GalCer and GlcCer) rendered VT1-unreactive Gb(3)/cholesterol vesicles, VT1-reactive. We found GalCer and GlcCer bind Gb(3), suggesting GSL-GSL interaction can counter cholesterol masking of Gb(3). The similar separation of Vero cell membrane-derived vesicles into minor "binding," and major "non-binding" fractions when probed with VT1, CTx, or anti-SSEA4 (a human GSL stem cell marker), demonstrates potential physiological relevance. Cell membrane GSL masking was cholesterol- and actin-dependent. Cholesterol depletion of Vero and HeLa cells enabled differential VT1B subunit labeling of "available" and "cholesterol-masked" plasma membrane Gb(3) pools by fluorescence microscopy. Thus, the model GSL/cholesterol vesicle studies predicted two distinct membrane GSL formats, which were demonstrated within the plasma membrane of cultured cells. Cholesterol masking of most cell membrane GSLs may impinge many GSL receptor functions.

Endothelial progenitor cells delivered into the pericardial space incorporate into areas of ischemic myocardium

Our objective was to determine whether autologous endothelial progenitor cells (EPCs) delivered into the pericardial space will migrate to and incorporate into ischemic myocardium in a porcine model. Use of EPCs to enhance neovascularization and preserve myocardial function in ischemic tissue is undergoing intense scrutiny as a potential therapy. Delivery into the pericardial sac may overcome some of the limitations of currently employed cell delivery techniques. EPCs were immunopurified from peripheral blood of Yorkshire pigs by selecting for the CD31 surface antigen, and adherent cells were cultured for 3-5 days. After myocardial ischemia was induced in the left anterior descending (LAD) artery, either autologous DiI (1,1'-dioctadecyl-1-3,3,3',3'-tetramethylindocarbocyanine perchlorate)-labeled EPCs (n=10) or serum-free medium (SFM; n=8) was delivered into the pericardial space using a percutaneous transatrial approach. Animals were sacrificed on Day 7 or 21. Echocardiography was performed at baseline, during ischemia, and on Day 7 in six SFM group animals and six EPC group animals. On Day 7, EPCs were identified in the left ventricular (LV) anterior wall or anterior septum in all six EPC-treated animals (cell density of 626 ± 122/mm(2)). On Day 21, EPCs were identified in the LV anterior wall or anterior septum in three of four EPC-treated animals (cell density of 267 ± 167/mm(2)). These cells showed dual staining for DiI and Bandeiraea simplicifolia lectin I (a marker of both native and exogenous endothelial cells). At the Day 7 follow-up, echocardiography demonstrated that fractional shortening in the EPC-treated group was 30.6 ± 3.4, compared with 22.6 ± 2.8 in SFM controls (P=.05). EPCs can migrate from the pericardial space to incorporate exclusively into areas of ischemic myocardium and may have favorable effects on LV function.

Bone Morphogenetic Protein Signaling Regulates Gastric Epithelial Cell Development and Proliferation in Mice

We investigated the role of bone morphogenetic protein (BMP) signaling in the regulation of gastric epithelial cell growth and differentiation by generating transgenic mice that express the BMP inhibitor noggin in the stomach. The promoter of the mouse H+/K+-ATPase β-subunit gene, which is specifically expressed in parietal cells, was used to regulate expression of noggin in the gastric epithelium of mice. The transgenic mice were analyzed for noggin expression, tissue morphology, cellular composition of the gastric mucosa, gastric acid content, and plasma levels of gastrin. Tissues were analyzed by immunohistochemical, quantitative real-time polymerase chain reaction, immunoblot, microtitration, and radioimmunoassay analyses. In the stomachs of the transgenic mice, phosphorylation of Smad 1, 5, and 8 decreased, indicating inhibition of BMP signaling. Mucosa were of increased height, with dilated glands, cystic structures, reduced numbers of parietal cells, and increased numbers of cells that coexpressed intrinsic factor, trefoil factor 2, and Griffonia (Bandeiraea) simplicifolia lectin II, compared with wild-type mice. In the transgenic mice, levels of the H+/K+-ATPase α-subunit protein and messenger RNA were reduced, whereas those of intrinsic factor increased. The transgenic mice were hypochloridric and had an increased number of Ki67- and proliferating cell nuclear antigen-positive cells; increased levels of plasma gastrin; increased expression of transforming growth factor-α, amphiregulin, and gastrin; and activation of extracellular signal-regulated kinase 2. Inhibiting BMP signaling in the stomachs of mice by expression of noggin causes loss of parietal cells, development of transitional cells that express markers of mucus neck and zymogenic lineages, and activation of proliferation. BMPs are therefore important regulators of gastric epithelial cell homeostasis.

Lectinhistochemical detection of terminal carbohydrate residues in the enteric myxozoan Enteromyxum leei parasitizing gilthead seabream Sparus aurata (Pisces: Teleostei): a study using light and transmission electron microscopy

The presence of terminal carbohydrate residues in Enteromyxum leei (Diamant, Lom et Dyková, 1994) Palenzuela, Redondo et Alvarez-Pellitero, 2002 stages in gilthead seabream intestines was studied at light microscopy (LM) and transmission electron microscopy (TEM) level using lectin histochemical techniques. Abundant mannose and/or glucose residues were demonstrated by the intense staining caused by binding of biotinylated concanavalin A (Con A), at both LM and TEM. A clear positivity was also obtained with Ulex europaeus (UEA I) agglutinin specific for fucose residues. Both lectins stained E. leei proliferative and sporogonic stages, though glycan patterns varied between these developmental stages. Wheat germ agglutinin (WGA) and Bandeiraea simplicifolia lectin I (BSL I) recognised only structures in the sporogonic stages. Faint labelling occurred with Glycine max (SBA) lectin. No staining was obtained with Sambucus nigra (SNA) agglutinin. The TEM studies demonstrated a restricted presence of N-acetyl-D-galactosamine and alpha-D-galactose, whereas glucose/mannose and fucose, the dominant structures, were also present at the parasite membranes and host-parasite interface, suggesting a role in host-parasite interaction.