A histochemical localization on Maclura pomifera lectin during osteogenesis

Mandibular condyles of 4-week-old Wistar strain rats and mandibles of ICR strain mice from 14 days gestation stage to 2 days postnatal stage were used to investigate the localization of Maclura pomifera lectin (MPA) during two modes of osteogenesis. During endochondral ossification of the mandibular condyle, MPA was only localized at the peripheral regions of calcified cartilage after the destruction of chondrocyte lacunae. Bone extracellular matrix (ECM) was not reacted with MPA. In intramembranous ossification of mice mandibles, MPA was stained intensively in the early bone ECM. The intensity of the MPA reaction decreased during bone development. In both cases of osteogenesis, chondroclasts and osteoclasts showed the strong affinity to MPA. These results indicated that the time- and position-specific changes within ECM proceeded during osteogenesis and that MPA was the useful probe to detect chondroclasts and osteoclasts.     

Cytotoxicity of lymphoid cells induced by Maclura pomifera (MP) lectin.

The cytotoxic activity of lymphoid cells stimulated with Maclura pomifera (MP) lectin was investigated. Spleen cells of Lewis (LEW) or Brown Norway (BN) rats induced a cell-dependent release of ⁵¹Cr from syngeneic, allogeneic, and xenogeneic erythrocytes when incubated with MP for 4–16 hr. The activity of MP differed from that of concanavalin A (Con A). MP exhibited a greater activity with spleen cells while Con A was more active when bone marrow cells were tested. Activity induced by MP required the presence of the lectin for at least 4 hr and was inhibited by melibiose, an inhibitor of MP binding. MP also stimulated phagocytosis by peritoneal macrophages of LEW rats, but phagocytosis was not responsible for the cytotoxic effect measured by ⁵¹Cr release. The ability of aggressor cells to bind MP did not correlate with their cytotoxic activity. The cytotoxic activity of spleen cells from athymic nude mice was equivalent to that of cells from euthymic littermates when stimulated with MP.     

Distribution of glycoconjugates localized by peanut and Maclura pomifera agglutinins during mouse molar root development.

Glycolipids, glycoproteins, glycosaminoglycans and sialoglycoproteins have all been implicated in a number of developmentally significant processes related to complex interactions between cell surfaces and the extracellular matrix. The present study was designed to localize glycoconjugates recognized by peanut agglutinin (PNA) and Maclura pomifera (MPA) lectins during mouse molar root development. Postnatal ICR mice at 10, 15, 21, 28 and 42 days were used. Lower jaws were dissected, fixed in 4% paraformaldehyde, decalcified in 5% EDTA and embedded in paraffin. Serial sections were made and stained with FITC-conjugated PNA or MPA. beta-Lactose was used as an inhibitory sugar for PNA, and alpha-D-melibiose for MPA. PNA specifically stained Hertwig's epithelial root sheath (HERS), whereas MPA stained a number of tissues. The outermost layer of root dentin, forming cellular cementum, alveolar bone and HERS showed positive reactions with MPA. Glycoconjugates localized by the lectins may be functionally related to molecules which contribute to root formation and cemento-genesis.     

Osage orange (Maclura pomifera): History and economic uses

Osage orange, a tree native to Texas, Oklahoma, and Arkansas, was planted widely in the United States as a living fence from 1850–1875. It has naturalized over much of its planted range and is an early successional species. Economically the tree has had a wide number of uses including for bows, yellow dye, and building materials. It is used for fence posts and is a potential source for a proteolytic enzyme. Its taxonomy, anatomy, morphology, chemical constitutents, medicinal uses, and toxicity are discussed in this paper and related to its economic importance.     

Phenylpropanoid glycosyltransferases from osage orange (Maclura pomifera) fruit

Flavonoids and isoflavonoids are well known for their beneficial effects on human health and their anti-insect and anti-microbial activities in plants. Osage orange fruit is rich in prenylated isoflavones and dihydrokaempferol and its glucoside. Four glycosyltransferases were identified from a collection of osage orange fruit expressed sequence tags. Biochemical characterization suggested that the glycosyltransferase UGT75L4 might be responsible for glucosylation of dihydrokaempferol in vivo, although this enzyme exhibited broad substrate recognition toward isoflavonoids and flavonoids in vitro. UGT88A4 was active on coumarin substrates. Identification of highly active phenylpropanoid glycosyltransferases will facilitate the metabolic engineering of glycosylated natural products in plants.     

The amino acid sequences of jacalin and the Maclura pomifera agglutinin

Amino acid sequences for the alpha-chains of the Moraceae lectins, jacalin and Maclura pomifera agglutinin, were determined by protein sequencing. Both are 133 residues long and contain several genetically variant positions; the overall homology is 85%. A possible site for the known glycopeptide of jacalin was located. The alpha-chains have a conserved tryptophan residue that may be part of the binding-site.     

Pomiferin, histone deacetylase inhibitor isolated from the fruits of Maclura pomifera

The major constituents from the fruits of Maclura pomifera are the prenylated isoflavones, osajin (1) and pomiferin (2). Their structures were elucidated using NMR spectroscopic techniques and mass spectrometric analysis. Compound 2 showed potential inhibitory activity in histone deacetylase (HDAC) enzyme assay. It also exhibited growth inhibitory activity on five human tumor cell lines and more sensitive inhibitory activity on the HCT-15 colon tumor cell line. Further structure-activity relationships of position 3 on ring B from aromatic ring will be reported in due course.     

The conformation of the T-antigen disaccharide bound to Maclura pomifera agglutinin in aqueous solution

The complex of Maclura pomifera agglutinin with the T-antigen disaccharide (beta-d-Gal-(1-->3)-alpha-d-GalNAc-(1-->O)-Me) was investigated by NMR spectroscopy in aqueous solution. Intramolecular transferred nuclear Overhauser enhancement (NOE) effects between the monosaccharide moieties were used to derive the ligand conformation in the lectin-bound state. Ligand protons in contact with the protein were identified by saturation transfer difference experiments and intermolecular transferred NOE effects. It is demonstrated that structural differences exist for the ligand-lectin complex in aqueous solution as compared with the previously published crystal structure (Lee, X., Thompson, A., Zhiming, Z., Ton-that, H., Biesterfeldt, J., Ogata, C., Xu, L., Johnston, R. A. Z. , and Young, N. M. (1998) J. Biol. Chem. 273, 6312-6318). In order to accommodate the O-methyl group of the disaccharide, the amino acid side chain of Tyr-122 has to rotate from its position in the crystal. The NMR data are in accord with two conformational families at the beta-(1-->3)glycosidic linkage in the solution complex with interglycosidic angles phi/psi = 45/-65 degrees and -65/-18 degrees. These differ from the bound conformation of the ligand in the crystal (phi/psi = 39/-8 degrees ) and are not highly populated by the ligand in the free state. The reason for the structural differences at the beta-(1-->3)glycosidic linkage are hydrogen bonds that stabilize the relative orientation of the monosaccharide units in the crystal. Our results demonstrate that the crystallization of a protein-carbohydrate complex can interfere with the delicate process of carbohydrate recognition in solution.     

The Fruits of Maclura pomifera Extracts Inhibits Glioma Stem-Like Cell Growth and Invasion

Glioma is the most common primary intracranial tumour. Recently, growing evidence showed that glioma possesses stem-like cells, which are thought to be chemo- and radio-resistant and believed to contribute to the poor clinical outcomes of these tumours. In this study, we found that stem-like glioma cells (CD133+) were significantly increased in neurosphere cells, which are highly invasive and resistant to multiple chemotherapeutic agents. From our natural products library, we screened 48 natural products and found one compound, Pomiferin, which was of particular interest. Our results showed that Pomiferin could inhibit cell viability, CD133+ cell population, sphere formation, and invasion ability of glioma neurosphere cells. We also found that multiple stemness-associated genes (BIM1, Nestin, and Nanog) were down-regulated by Pomiferin treatment of glioma neurosphere cells. Taken together, our results suggest that Pomiferin could kill the cancer stem-like cells in glioma and may serve as a potential therapeutic agent in the future.     

Identification and characterization of the pulmonary alveolar type II cell Maclura pomifera agglutinin-binding membrane glycoprotein

The lectin Maclura pomifera agglutinin (MPA) binds to the apical surface of pulmonary alveolar type II but not type I cells. We show that MPA binds to a single membrane glycoprotein in type II cells with a molecular mass of 230 kDa in the rabbit and 200 kDa in the rat. The glycoprotein has an abundance of terminal N-acetylgalactosamine residues. It is a hydrophilic integral membrane protein suggesting that it has an extensive extramembrane domain or is an ion channel. The glycoprotein is similar in rat and rabbit, with the exception that the rat glycoprotein is partially sialylated and is trypsin sensitive. The MPA-binding glycoprotein represents a new integral membrane marker of the apical domain of the pulmonary alveolar type II cell.     

Heparin binding lectin of human placenta as a tool for histochemical ligand localization and isolation

Biotinylated heparin has been used to detect the presence of specific binding sites in sections of human placenta, which has prompted demonstration of expression of lectin activity for this proteoglycan. Purification of this lectin from full-term placenta facilitates the synthesis of its biotinylated derivative, using biotin-amidocaproyl hydrazide, without affecting its activity. It also enables immunization to obtain antibodies. The labeled lectin is shown to bind specifically to nuclear and cytoplasmic locations in various cell types of human placenta, nuclear expression of lectin binding sites being more pronounced at the full-term stage than after 8 weeks of development. The structurally related histone H2B exhibits obvious differences in its binding pattern. The presence of ligands accessible to the lectin whose binding activity can be inhibited by addition of an excess of heparin correlates in most instances with the level of lectin expression detected immunohistochemically. Biochemical information on the nature of the glycohistochemically inferred lectin-specific ligand(s) is obtained by affinity chromatography on resin-immobilized lectin. It leads to isolation of a proteoglycan with similar electrophoretic mobility in agarose-polyacrylamide gel electrophoresis relative to the independently purified heparan sulfate-containing fibronectin binding proteoglycan from human placenta. Both fractions inhibit binding of heparin to the lectin and contain immunologically detected co-purified lectin, emphasizing their ligand properties. Application of labeled tissue lectins in conjunction with lectin-specific antibodies is proposed to obtain valuable insights into the expression of the receptor as well as the ligand part of protein-carbohydrate recognition.     

A lectin histochemical study of the oesophagus of shi drum

The saccharide composition of surface and secretion glycoconjugates in the oesophagus of Umbrina cirrosa was examined by means of lectin histochemistry. Mucous cells showed the presence of N‐acetylgalactosamine, N‐acetylglucosamine and sialic acid linked to the dimer galactosyl(β1→3) N‐acetylgalactosamine. Columnar epithelial cells had a positive reaction with almost all the lectins employed, located in the supranuclear region and in the cell coat. The presence of abundant and various glycoconjugates in the secretions of shi drum oesophagus was correlated to the absence of salivary glands in fishes in general.     

Homology of the D-galactose-specific lectins from Artocarpus integrifolia and Maclura pomifera and the role of an unusual small polypeptide subunit

The Maclura pomifera agglutinin (MPA) was purified by affinity chromatography from a seed extract and its properties were compared with those of the Artocarpus integrifolia lectin, jacalin. Reverse-phase high-performance liquid chromatography showed both proteins had multiple forms of a small approximately 20-residue polypeptide chain in addition to the major 12,000 Mr subunit. The amino acid sequences of the small chains and the N-terminal sequences of the large subunits showed considerable similarity between the two proteins, approximately 60% identical residues. The homology of the proteins was confirmed by the similarity of their circular dichroism and fluorescence emission spectra. MPA showed much greater spectral changes upon binding methyl alpha-D-galactoside, suggesting it has complete activity rather than the partial activity found for jacalin. The binding of methyl alpha-D-galactoside by MPA was measured by fluorescence titration; the KA was 1.9 X 10(4) M-1 compared to 3.4 X 10(4) M-1 for jacalin. MPA also precipitated human IgA1 in the same manner as jacalin. The spectra indicate the involvement of tryptophan and tyrosine residues in the binding site of these lectins. Since a tryptophan residue is conserved in all the small subunits, they may form part of the binding site.     

Glycoconjugate localization in larval and adult skin of the bullfrog, Rana catesbeiana: a lectin histochemical study

This study investigates whether or not the distribution of specific glycoconjugates within the skin is related to the regulation of water balance in the aquatic larvae and semiaquatic adults of the bullfrog, Rana catesbeiana. A lectin histochemical study was carried out on paraffin sections of dorsal and ventral skin from tadpoles in representative stages as well as from adult frogs. Sections were stained with the following horseradish peroxidase (HRP)-conjugated lectins, which bind to specific terminal sugar residues of glycoconjugates: UEA 1 for alpha-L-fucose, SBA for N-acetyl-D-galactosamine, WGA for N-acetyl-B-D-glucosamine, and PNA for beta-galactose. Results indicate that lectins serve as markers for specific skin components (e.g., a second ground substance layer within the dermis was revealed by positive UEA 1 staining). Moreover, each lectin has a specific binding pattern that is similar in dorsal and ventral skin; the larval patterns change as the skin undergoes extensive histological and physiological remodeling during metamorphic climax. These findings enhance our understanding of glycoconjugates and their relationship to skin structure and function-in particular, to the regulation of water balance in R. catesbeiana.     

Immunohistochemical localization of bone morphogenetic protein-signaling Smads during long-bone distraction osteogenesis.

In this study we investigated the expression of bone morphogenetic protein (BMP)-signaling Smads in distraction osteogenesis (DO). Osteotomy of the right tibia was performed in 14 skeletally mature white New Zealand male rabbits. Lengthening was started 1 week later at a rate of 0.5 mm/12 hr and was maintained for 3 weeks. Expression of Smad proteins 1, 4, 5, 6, 7, and 8 and Smad ubiquitin regulatory factors (Smurfs) 1 and 2 was evaluated in the distracted zone using immunohistochemistry. Expression of receptor-regulated Smads (R-Smads) 1, 5, and 8 showed a significant increase during the distraction phase, followed by a gradual decrease during the consolidation phase. Smad 4 showed significant expression during both distraction and the beginning of the consolidation phase. Smad 6 and Smad 7 were highly expressed during the consolidation phase. Staining for both Smurfs 1 and 2 was maximal at the end of the distraction period. Staining for all proteins was most intense in chondrocyte and fibroblast-like cells. Expression pattern of R-Smads correlated with our previously reported expression pattern of BMPs 2, 4, and 7 and their receptors. These results therefore suggest a role for the whole BMP signaling pathway including the Smad proteins in DO.