Differential expression of glycans in the urothelial layers of horse urinary bladder.

BACKGROUND: Urothelium is a multilayer epithelium covering the inner surface of the urinary bladder that acts as a blood-urine barrier and is involved in maintaining the wellbeing of the whole organism. Glycans serve in the maturation and differentiation of cells and thus play a key role in the morphology and function of the multilayered epithelium. The aim of the present study was to examine the glycoprotein pattern of the horse urinary bladder urothelium by lectin histochemistry. METHODS: The study involved urinary bladders from four horse stallions. Tissue sections were stained with a panel of eleven lectins, in combination with saponification and sialidase digestion (Ks). RESULTS: Basal cells displayed high-mannose N-glycans (Con A), α2,6-linked sialic acid (SNA), and O-linked sialoglycans with sialic acids linked to Galßl,3GalNAc (T antigen) (KsPNA) and terminal N-acetylgalactosamine (Tn antigen) (KsSBA). The young intermediate cells expressed terminal N-acetylglucosamine (GlcNAc) (GSA II), galactose (GSA I-B4), T- and Tn antigens (PNA, SBA). The mature intermediate cells showed additional high-mannose N-glycans, O-linked sialoglycans (sialyl-T antigen, sialyl-Tn antigen), α2,6- and α2,3-linked sialic acid (MAL II), α1,2-linked fucose (UEA I), and GlcNAc (KsWGA). The latter residue marked the boundary with the overlying surface layer. Few Con A positive intermediate cells were seen to cross the entire urothelium thickness. The surface cells showed additional glycans such as T antigen and sialic acids linked to GalNAc binding DBA (KsDBA). Few surface cells contained α1,3-linked fucose (LTA), whereas some other cells displayed intraluminal secretion of mucin-type glycans terminating with GalNAcα1,3(LFucα1,2)Galß1,3/4GlcNAcß1 (DBA). The luminal surface expressed the most complex glycan pattern in the urothelium because only α1,3-linked fucose lacked among the demonstrated glycans. CONCLUSIONS: This study showed that the glycan pattern becomes more complex from the basal to surface layer of the urothelium and that surface cells could modify the composition of urine via the secretion of glycoproteins.

Ultrastructural characteristics of ovine bone marrow-derived mesenchymal stromal cells cultured with a silicon stabilized tricalcium phosphate bioceramic.

Bioceramics are being used in experimental bone engineering application in association with bone marrow derived mesenchymal stem cells (BM-MSCs) as a new therapeutic tool, but their effects on the ultrastructure of BM-MSCs are yet unknown. In this study we report the morphological features of ovine (o)BM-MSCs cultured with Skelite, a resorbable bioceramic based on silicon stabilized tricalcium phosphate (SiTCP), able to promote the repair of induced bone defect in sheep model. oBM-MSCs were isolated from the iliac crest, cultured until they reached near-confluence and incubated with SiTCP. After 48 hr the monolayers were highly damaged and only few cells adhered to the plastic. Thus, SiTCP was removed, and after washing the cells were cultured until they became confluent. Then, they were trypsinizated and processed for transmission electron microscopy (TEM) and RT-PCR analysis. RT-PCR displayed that oBM-MSCs express typical surface marker for MSCs. TEM revealed the presence of electron-lucent cells and electron-dense cells, both expressing the CD90 surface antigen. The prominent feature of electron-lucent cells was the concentration of cytoplasmic organelles around the nucleus as well as large surface blebs containing glycogen or profiles of endoplasmic reticulum. The dark cells had a multilocular appearance by the presence of peripheral vacuoles. Some dark cells contained endocytic vesicles, lysosomes, and glycogen aggregates. oBM-MSCs showed different types of specialized interconnections. The comparison with ultrastructural features of untreated oBM-MSCs suggests the light and dark cells are two distinct cell types which were differently affected by SiTCP bioceramic. Skelite cultured ovine BM-MSCs display electron-dense and electron-lucent cells which are differently affected by this bioceramic. This suggests that they could play a different role in bioceramic based therapy.

Ultrastructural study of cultured ovine bone marrow-derived mesenchymal stromal cells.

Ovine bone marrow-derived mesenchymal stromal cells (oBM-MSCs) represent a good animal model for cell-based therapy and tissue engineering. Despite their use as a new therapeutic tool for several clinical applications, the morphological features of oBM-MSCs are yet unknown. Therefore, in this study the ultrastructural phenotype of these cells was analysed by transmission electron microscopy (TEM). The oBM-MSCs were isolated from the iliac crest and cultured until they reached near-confluence. After trypsinization, they were processed to investigate their ultrastructural features as well as specific surface marker proteins by flow cytometry and immunogold electron microscopy. Flow cytometry displayed that all oBM-MSCs lacked expression of CD31, CD34, CD45, HLA-DR whereas they expressed CD44, CD58, HLAI and a minor subset of the cell population (12%) exhibited CD90. TEM revealed the presence of two morphologically distinct cell types: cuboidal electron-lucent cells and spindle-shaped electron-dense cells, both expressing the CD90 antigen. Most of the electron-lucent cells showed glycogen aggregates, dilated cisternae of RER, moderately developed Golgi complex, and secretory activity. The electron-dense cell type was constituted by two different cell-populations: type A cells with numerous endosomes, dense bodies, rod-shaped mitochondria and filopodia; type B cells with elongated mitochondria, thin pseudopodia and cytoplasmic connectivity with electron-lucent cells. These morphological findings could provide a useful support to identify "in situ" the cellular components involved in the cell-therapy when cultured oBM-MSCs are injected.

Effects of mefepronic acid (2-phenoxy-2-methyl propionic acid) on hepatic metabolism and reproductive parameters in postpartum dairy cows.

This study investigates the effects of mefepronic acid (MA), a PPAR-α agonist, on hepatic metabolic functions and reproduction of postpartum dairy cows. Sixty Friesian cows were divided into Group A (administered 5g of MA IM, within 24 hrs after calving, on the 3(rd) and 5(th) day postpartum) and Group B (control). All the cows were blood sampled within 24 hrs of calving (Day 0), on Day 3, 5, 10, 15, 30, and 40 postpartum. On plasma, metabolic and biochemical parameters were determined. Liver biopsies were performed on Day 0, 15 and 30 for the evaluation of hepatic lipid and glycogen content. Reproductive parameters were also evaluated. In Group A, blood HDL, glucose and cholesterol increased till the end of the study, in accordance with the histological results. PPAR-α immunopositive cells increased in liver slices of Group A, too. Reproductive parameters improved in Group A. This study highlights the beneficial effects of mefepronic acid on the hepatic metabolism and reproductive parameters of post-partum dairy cows.

In situ characterization of glycans in the urothelium of donkey bladder: evidence of secretion of sialomucins.

The glycoprotein pattern was investigated by lectin histochemistry in the urothelium lining the urinary bladder of the donkey Equus asinus. Tissue sections were stained with a panel of twelve lectins, in combination with saponification and sialidase digestion (K-s). The urinary bladder urothelium has three distinct layers from the basal zone to the lumen consisting of basal, intermediate and superficial cells (umbrella cells). Cytoplasm of basal cells reacted with SNA, PNA, K-s-PNA, GSA I-B4 and Con A showing glycans ending with Neu5Acα2,6Gal/GalNAc, Neu5AcGalß1,3GalNAc, αGal and with terminal/internal αMan. The cytoplasm of umbrella cells displayed an increase of Neu5AcGalß1,3GalNAc and the appearance of Neu5AcGalß1,3GalNAc, Neu5acα2,3Galß1,4GlcNAc and Neu5AcGalNAc residues (MAL II, K-s-SBA and K-s-HPA staining). Scattered umbrella cells were characterized by glycans terminating with GalNAc binding DBA, SBA and HPA. The mucosa forms folds with a crypt-like appearance where the urothelium shows a different pattern of glycans. The bladder luminal surface stained with K-s-PNA, K-s-DBA, KOH-s-SBA, and K-s-HPA displaying a coating of sialoglycoproteins belonging to O-linked glycans (typical secretory moieties). These findings show that different glycosylation patterns exist along the donkey bladder urothelium, and different sub-populations of umbrella cells are present secreting the sialoglycans which constitute the protective gel layer lining the bladder.

Lectin-binding pattern of the Senegalese sole Solea senegalensis oogenesis.

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH-sialidase (K-s)-WGA, GSA I-B(4) in the late vitellogenic stage, and in wild fish also SNA and K-s-PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I-B(4) only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B(4) in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA(120) during the late vitellogenic stage, also SNA, PNA, K-s-PNA, GSA I-B(4) in cultured fish, DBA, and K-s-WGA in wild ones which stained with SBA, HPA, and GSA I-B(4) in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K-s-PNA, SBA, HPA, K-s-WGA, GSA I-B(4), UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I-B(4), GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I-B(4) reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture-rearing conditions.

Morphometric features and glycoconjugate pattern of rabbit intestine are affected by particle size of pelleted diets.

Feed particle size effects on morphology and glycoconjugate pattern was investigated in the rabbit intestine. Rabbits fed with fine particles (2 mm) displayed more irregularly shaped, higher duodenal villi and deeper crypts in distal colon as well as higher number of goblet cells than coarse (8 mm) fed ones. Brush border expressed: (i) in duodenum, neutral/sulfated glycoconjugates and glycans binding MAL II, SNA, Con A than KOH-sialidase-PNA and DBA reactivity in fine and coarse fed rabbits, respectively, (ii) in cecum, mainly sulfoglycans in coarse fed rabbits, MAL II and PNA staining in all samples, and (iii) in distal colon few sulfoglycans and MAL II reactivity. Enterocytes bound MAL II in duodenum, Con A in cecum, DBA, and Con A in distal colon of all rabbits, SNA in distal colon of coarse fed ones. Brunner's glands displayed high presence of acidic/sulfated mucins in fine fed rabbits, neutral glycoconjugates and reactivity with MAL II, SNA, PNA, KOH-sialidase-PNA, and Con A in all rabbits. Goblet cells exhibited: (i) in duodenum neutral and sulfomucins as well as MAL II and KOH-sialidase-PNA staining, than SNA and DBA in fine and coarse fed rabbits, respectively, (ii) in cecum sulfated glycans, MAL II, SNA, KOH-sialidase-PNA, DBA reactivity, and (iii) in distal colon acidic/sulfomucins, MAL II and SNA staining, and DBA reactivity in fine fed specimens. Crypt cells exhibited neutral and PNA reactive glycoconjugates in the cecum. In the distal colon also acidic/sulfated glycans, and MAL II, KOH-sialidase-PNA, DBA; SNA staining showed weaker reactivity in fine fed rabbits, which bound Con A.

Morphology and lectin-binding sites of pyloric caeca epithelium in normal and GnRH-treated Atlantic bluefin tuna (Thunnus thynnus) Linnaeus 1758.

Mucosal epithelium of pyloric caeca was studied in normal and in GnRH-treated Atlantic bluefin tuna Thunnus thynnus L., using morphological analysis, conventional and lectin glycohistochemistry. The lining epithelium consisted of columnar (absorptive) cells, goblet cells and intraepithelial leucocytes. The epithelium from normal animals was significantly taller than GnRH-treated samples. Conventional histochemistry displayed the same staining pattern in normal and hormone-treated specimens which showed a mixture of neutral and sulphated acidic glycoconjugates in the luminal surface and goblet cells, and neutral glycans in apical granules of enterocytes. Lectin histochemistry revealed a different glycoconjugate pattern in normal and GnRH-treated tunas. In normal specimens the luminal surface expressed sialoglycoconjugates which bound MAL II, SNA, KOH-sialidase-PNA, KOH-sialidase-SBA as well as asialoglycans stained with HPA, SBA, GSA I-B4 , LTA. N-linked glycans were highlighted by Con A and KOH-sialidase-WGA. In GnRH-treated tunas the luminal surface did not react with SNA, SBA and LTA. The columnar cells of normal tunas bound KOH-sialisase-PNA in the apical region, KOH-sialidase-PNA, KOH-sialidase-DBA, HPA, SBA, KOH-sialidase-SBA and KOH-sialidase-WGA in apical granules, GSA I-B4 and LTA in the supranuclear region. GnRH-treated specimens showed some columnar cells that stained with KOH-sialidase-WGA in the apical granules and with GSA I-B4 in the supranuclear region. The goblet cells of normal animals produced mucins positive to PNA, HPA, KOH-sialidase-DBA, SBA, GSA II. The latter three binding sites lacked in GnRH-treated tunas. The results suggest that the mucosal epithelium of Thunnus thynnus L. pyloric caeca expresses a complex glycan pattern that is affected by GnRH-treatment.

Performance, gut morphology and carcass characteristics of fattening rabbits as affected by particle size of pelleted diets.

A review of past literature revealed inconsistencies in recommended feed particle size for optimal growth and productive performance of rabbits. Changing diet formulation and subsequent processing conditions may improve pellet texture and potentially affect rabbit performance. In the current study, two isoenergetic and isonitrogenous pelleted diets were formulated, which varied in the particle size of the concentrates (2 and 8 mm, respectively). The objective was to evaluate the effect of different particle sizes of compound diets on performance, nutrient utilisation, gut morphology, and carcass characteristics of fattening Italian White breed rabbits. The finely ground diet led to a significant improvement in feed efficiency and apparent digestibility of crude protein, ether extract, crude fibre and NDF, without any negative effect on gut morphology. Furthermore, a smaller particle size of concentrates in pelleted diets improved carcass traits. Meat colour parameters showed significant differences in longissimus lumborum and biceps femoris due to dietary treatments, but in both muscles pH values 1 h and 24 h after slaughter remained unchanged. It is concluded that a finely ground pelleted diet can be used to improve growth performance of rabbits without affecting carcass parameters.

Lectin histochemistry of gastrointestinal glycoconjugates in the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774).

Mucins in the gastrointestinal tract of Rhinolophus ferrumequinum were investigated by histochemistry and lectin histochemistry to evaluate morphofunctional variations of different regions and their possible physiological and evolutionary implications. Histochemical methods included periodic acid-Schiff (PAS), Alcian blue (AB) at pH 2.5 and 1.0 and high-iron-diamine AB pH 2.5. Binding of lectins Con A, DBA, WGA, LTA, LFA, PNA and SBA; LFA, PNA and SBA with prior sialidase treatment; and paradoxical Con A were evaluated. The oesophagus lacked glands. The stomach was divided into a short cardias, a wide fundus and a brief pylorus. The surface muciparous cells secreted sulpho- and sialomucins with N-acetylgalactosamine (GalNAc) residues, N-acetyllactosamine and (beta1,4 N-acetylglucosamine)(n) chains. Towards the pylorus, N-acetylgalactosamine residues disappeared and acidity decreased. Cardiac glands, neck cells in the fundic glands, pyloric and duodenal Brunner's glands all shared neutral, stable class-III mucins, mainly with N-acetylgalactosamine sequences. The intestine was divided into a duodenum, a jejuno-ileum and a short rectum. The goblet cells produced sulpho- and sialomucins with sialylated N-acetylgalactosamine sequences, (beta1,4 N-acetylglucosamine)(n) and N-acetyllactosamine, whose sialylation increased towards the rectum. The main features of the mucins are probably associated with the requirements of fast absorption and food passage and in protection against mechanical and pathogenic injuries.

Glycoconjugate histochemistry of the digestive tract of Triturus carnifex (Amphibia, Caudata).

In this study, the variety of sugar residues in the gut glycoconjugates of Triturus carnifex (Amphibia, Caudata) are investigated by carbohydrate conventional histochemistry and lectin histochemistry. The oesophageal surface mucous cells contained acidic glycoconjugates, with residues of GalNAc, Gal beta1,3 GalNAc and (GlcNAc beta1,4)(n) oligomers. The gastric surface cells mainly produced neutral glycoproteins with residues of fucose, Gal beta1-3 GalNAc, Gal-alphaGal, and (GlcNAc beta1,4)(n) oligomers in N- and O-linked glycans, as the glandular mucous neck cells, with residues of mannose/glucose, GalNAc, Gal beta1,3 GalNAc, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,6 or terminal alpha1,3 or alpha1,4 in O-linked glycans. The oxynticopeptic tubulo-vesicular system contained neutral glycoproteins with N- and O-linked glycans with residues of Gal-alphaGal, Gal beta1-3 GalNAc and (GlcNAc beta1,4)(n)oligomers; Fuc linked alpha1,2 to Gal, alpha1,3 to GlcNAc in (poly)lactosamine chains and alpha1,6 to GlcNAc in N-linked glycans. Most of these glycoproteins probably corresponds to the H(+)K(+)-ATPase beta-subunit. The intestinal goblet cells contained acidic glycoconjugates, with residues of GalNAc, mannose/ glucose, (GlcNAc beta1,4)(n)oligomers and fucose linked alpha1,2 to Gal in O-linked oligosaccharides. The different composition of the mucus in the digestive tracts may be correlated with its different functions. In fact the presence of abundant sulphation of glycoconjugates, mainly in the oesophagus and intestine, probably confers resistance to bacterial enzymatic degradation of the mucus barrier.

Pepsinogen and H,K-ATPase mediate acid secretion in gastric glands of Triturus carnifex (Amphibia, Caudata).

The gastric glands of Triturus carnifex (Amphibia, Caudata) have been examined by histochemical and immunohistochemical methods with particular regard to hydrochloric acid and pepsinogen secretion. Fundic glands consist of mucous neck cells, endocrine cells and oxynticopeptic cells producing both pepsinogen and hydrochloric acid. The neck cells showed an unexpected distribution pattern which was only observed in the oral fundus, and produced neutral mucins with glycosidic residues of GalNAc and Gal beta1,3GalNAc, and in this respect they differ from the neck cells of anuran amphibians. The secretion of pepsinogen and hydrochloric acid as demonstrated by immunolabelling with anti-H,K-ATPase and with anti-pepsinogen, respectively, seems not to vary significantly along the longitudinal axis of the stomach. The mechanism of gastric acid secretion seems to be mediated by an ATPase, having similar features to the mammalian gastric H,K-ATPase, and is localised in the luminal membrane and in the subapical cytoplasm of the oxynticopeptic cells. Unusually, the same cytoplasmic areas revealed binding specificity for the winged pea lectin (WPA) from Lotus tetragonolobus, even after beta elimination, indicating the presence of fucosyl residues in N-linked oligosaccharidic chains in glycoproteins of beta-H,K-ATPase subunits.