Altered glycosylation in secreting cells of the gastric glands of aquaporin-4-deficient mice.

Aquaporins (AQPs) are important for water transport in the gastrointestinal tract. Changes in their expression and/or localization could cause in disorders and be used as therapeutic targets. Aquaporin-4 (AQP4) is expressed predominantly on the basolateral membrane of the parietal cells in the corpus of the murine gastric glands. Although the secretion of gastric juice is not affected in AQP4-deficient knockout, we evaluated by light microscopy whether the lack of AQP4 affects the glycopatterns of secreting gastric cells. Wild type (WT) and AQP4-deficient knockout mice (KO) were fed a standard diet ad libitum before sacrifice. Segments of stomach corpus were collected, fixed in buffered formalin, and embedded in paraffin wax. Sections, 5-µm thick, were analyzed by histochemical methods (Periodic acid-Schiff, Alcian Blue pH 2.5), and binding of lectins specific to GalNAc (SBA, DBA), Gal (PNA) GlcNAc (WGA, GSAII) mannose and/or glucose (ConA), and fucose (UEA-I, AAA, LTA). Immunohistochemical methods such as anti-Muc6 for neck cells and anti- ß- H+/K+-ATPase for parietal cells were also performed. Compared to WT mice, in the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated, and fucosylated residues were observed; lower fucosylation resulted also in the parietal cells. The observed differences of KO in respect to WT could lead to severer pathological conditions. RESEARCH HIGHLIGHTS: Glycopatterns in gastric glands were compared between wild type (WT) and AQP4-deficient knockout (KO) mice by histochemical and lectin-binding methods. In the mucous cells of KO lower amounts of glycans with galactosyl/galactosaminylated, glycosyl/glycosaminylated and fucosylated residues were observed. In the parietal cells lower fucosylation also resulted. AQP4-deficiency affects glycosylation and could result in altered functionality and pathological conditions.

Aluminum exposure alters the pedal mucous secretions of the chocolate-band snail, Eobania vermiculata (Gastropoda: Helicidae).

Aluminum (Al) is used in everyday life and present in food drugs, packaging, industry, and agriculture. Although it is the most common metal in the Earth crust, a correlation has been demonstrated between its presence and various pathologies, even serious ones, especially of a neurological type. However, there is a histological gap regarding the role Al can have in contact with the covering and secreting epithelia. The alterations of the ventral and dorsal foot mucocytes and their secretions of the snail Eobania vermiculata caused by Al were investigated in situ by histochemical and lectin-histochemical techniques. Administration to different experimental groups took place for 3 and 9 days with 50 and 200 µM of AlCl3. Several types of mucocytes were detected with a prevalent secretion of acid glycans in the foot of E. vermiculata. Sulfated glycans prevail in the dorsal region, with one type showing only fucosylated residues and another also having galactosaminylated and glycosaminylated residues. Carboxylated glycans prevail in the ventral region, with presence of galactosaminylated, glycosaminylated, and fucosylated residuals in both cells. Snails treated presented a general decrease of mucin amount in the secreting cells and affected the mucus composition. These changes could alter the rheological and functional properties of the mucus with possible implications for the health of the treated animals. RESEARCH HIGHLIGHTS: Snails were fed with Al-contaminated lettuce at different concentrations. In the foot mucocytes produced mucus with prevailing acidic glycans. In the treated resulted a reduction in the amount of mucus and an alteration of glycan composition.

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.

Sexual and asexual reproduction in a Mediterranean Tethya (Porifera, Demospongiae) species.

BACKGROUND: The reproductive cycle of the recently described sponge Tethya meloni was investigated for a period of 15 months (September 2018 - November 2019) in the Mar Piccolo of Taranto (Southern Italy) and was compared with data previously collected for the other two sympatric species of the same genus known for Mediterranean Sea, T. citrina and T. aurantium. RESULTS: T. meloni is a gonochoric species with a sex ratio strongly shifted towards females. Asexual budding was a seasonal process, limited to few specimens. In a specimen collected in September 2018 both oocytes and buds occurred, suggesting that in T. meloni the sexual and asexual phases may coexist both at the population and individual levels. CONCLUSIONS: The data obtained from this research compared with the available literature confirm the high temporal variability of the reproductive cycles in the Mediterranean species of Tethya, but with common general characteristics. In sexual reproduction, the oocyte production period lasts several months, with a peak between summer and autumn while spermatogenesis, shorter but with greater reproductive effort, follows the onset of oogenesis. The asexual reproduction phase of T. meloni, on the other hand, occurs in a short period and seems to have less importance in the overall reproductive process.

Sweet as honey, bitter as bile: Mitochondriotoxic peptides and other therapeutic proteins isolated from animal tissues, for dealing with mitochondrial apoptosis.

Mitochondria are subcellular organelles involved in cell metabolism and cell life-cycle. Their role in apoptosis regulation makes them an interesting target of new drugs for dealing with cancer or rare diseases. Several peptides and proteins isolated from animal and plant sources are known for their therapeutic properties and have been tested on cancer cell-lines and xenograft murine models, highlighting their ability in inducing cell-death by triggering mitochondrial apoptosis. Some of those molecules have been even approved as drugs. Conversely, many other bioactive compounds are still under investigation for their proapoptotic properties. In this review we report about a group of peptides, isolated from animal venoms, with potential therapeutic properties related to their ability in triggering mitochondrial apoptosis. This class of compounds is known with different names, such as mitochondriotoxins or mitocans.

High-fat Diet Alters the Glycosylation Patterns of Duodenal Mucins in a Murine Model.

High-fat diet (HFD) alters the glycosylation patterns of intestinal mucins leading to several health problems. We studied by histochemical and lectin-binding methods mucin alterations in the duodenum of mice fed a HFD for 25 weeks. Histochemical methods included periodic acid-Schiff, alcian blue pH 2.5, and high-iron diamine. Lectin-binding experiments were performed with SBA, PNA, WGA, MAA-II, SNA, ConA, UEA-I, LTA, and AAA. SBA, PNA, WGA, MAA-II, and SNA were tested also after desulfation and ConA after periodate-sodium borohydrate treatments (paradoxical ConA). Duodenal mucins are secreted by Brunner's glands and goblet cells in the villi. Brunner's glands of HFD mice showed increased secreting activity and a general reduction of glycosylated residuals, such as fucose and terminal α1,4-linked GlcNAc. Moreover, a general reduction of glycosylated residuals in the goblet cells of villi such as the fucosylated and sulfated ones was observed. Since the cited residuals are involved in cytoprotective and cytostatic functions, as well as in interactions with the intestinal microbiota and protection against parasites and inflammatory disorders, we conclude that HFD can predispose duodenum to several possible health disorders.

Human Ovarian Cancer Tissue Exhibits Increase of Mitochondrial Biogenesis and Cristae Remodeling.

Ovarian cancer (OC) is the most lethal gynecologic cancer characterized by an elevated apoptosis resistance that, potentially, leads to chemo-resistance in the recurrent disease. Mitochondrial oxidative phosphorylation was found altered in OC, and mitochondria were proposed as a target for therapy. Molecular evidence suggests that the deregulation of mitochondrial biogenesis, morphology, dynamics, and apoptosis is involved in carcinogenesis. However, these mitochondrial processes remain to be investigated in OC. Eighteen controls and 16 OC tissues (serous and mucinous) were collected. Enzymatic activities were performed spectrophotometrically, mitochondrial DNA (mtDNA) content was measured by real-time-PCR, protein levels were determined by Western blotting, and mitochondrial number and structure were measured by electron microscopy. Statistical analysis was performed using Student's t-test, Mann-Whitney U test, and principal component analysis (PCA). We found, in OC, that increased mitochondrial number associated with increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and mitochondrial transcription factor A (TFAM) protein levels, as well as mtDNA content. The OC mitochondria presented an increased maximum length, as well as reduced cristae width and junction diameter, associated with increased optic atrophy 1 protein (OPA1) and prohibitin 2 (PHB2) protein levels. In addition, in OC tissues, augmented cAMP and sirtuin 3 (SIRT3) protein levels were observed. PCA of the 25 analyzed biochemical parameters classified OC patients in a distinct group from controls. We highlight a "mitochondrial signature" in OC that could result from cooperation of the cAMP pathway with the SIRT3, OPA1, and PHB2 proteins.

Comparative histochemical analysis of intestinal glycoconjugates in the blunthead pufferfish Sphoeroides pachygaster and grey triggerfish Balistes capriscus (Teleostei: Tetraodontiformes).

The localization of intestinal glycoconjugates of the blunthead pufferfish Sphoeroides pachygaster and the grey triggerfish Balistes capriscus from the north-western Ionian Sea was analysed by histochemical methods (PAS, AB pH 2.5, HID) and lectin binding experiments (WGA, LFA, SBA, sialidase-SBA, PNA, sialidase-PNA, ConA, AAA, UEA-I, LTA) to assess how evolutionary loss of a functional stomach in S. pachygaster affects intestinal secretions relative to the B. capriscus, which retains the plesiomorphic gastric condition. Sphoeroides pachygaster had a lower content of acid mucins but more complex sialylation patterns than B. capriscus. GalNAc and GlcNAc residuals were present in both, but GalNAc residuals in S. pachygaster were subterminal to sialic acid. Balistes capriscus lacked galactosylated residuals and its enterocytes had a glycocalyx that differed in composition between the small intestine and the rectum and was missing from S. pachygaster. Functional and ecological implications of these findings are discussed.

Multilocus phylogeny and coalescent species delimitation in Kotschy's gecko, Mediodactylus kotschyi: Hidden diversity and cryptic species.

Kotschy's Gecko, Mediodactylus kotschyi, is a small gecko native to southeastern Europe and the Levant. It displays great morphological variation with a large number of morphologically recognized subspecies. However, it has been suggested that it constitutes a species complex of several yet unrecognized species. In this study, we used multilocus sequence data (three mitochondrial and three nuclear gene fragments) to estimate the phylogenetic relationships of 174 specimens from 129 sampling localities, covering a substantial part of the distribution range of the species. Our results revealed high genetic diversity of M. kotschyi populations and contributed to our knowledge about the phylogenetic relationships and the estimation of the divergence times between them. Diversification within M. kotschyi began approximately 15 million years ago (Mya) in the Middle Miocene, whereas the diversification within most of the major clades have been occurred in the last 5 Mya. Species delimitation analysis suggests there exists five species within the complex, and we propose to tentatively recognize the following taxa as full species: M. kotschyi (mainland Balkans, most of Aegean islands, and Italy), M. orientalis (Levant, Cyprus, southern Anatolia, and south-eastern Aegean islands), M. danilewskii (Black Sea region and south-western Anatolia), M. bartoni (Crete), and M. oertzeni (southern Dodecanese Islands). This newly recognized diversity underlines the complex biogeographical history of the Eastern Mediterranean region.

Characterization of the skin mucus in the common octopus Octopus vulgaris (Cuvier) reared paralarvae.

The Octopus vulgaris farming is impaired by the high mortality of the paralarvae during the first month of life. Several factors have been investigated in this regard, but no data exist on the body surface mucus, which represents the interface with the outside environment. This study included morphometric analysis and glycoconjugates characterization of skin mucus in reared Octopus vulgaris paralarvae during the first month of life. Four types of mucous cells were distinguished:  mucous 1 (m1) and mucous 2 (m2) cells were scattered in the mantle epidermis, mucous 3 (m3) and mucous 4 (m4) in the epithelium surrounding the sucker. Except for the presence of fucosylated and neutral glycoconjugates in all mucous cells, each cell type expressed a characteristic glycopattern. m2 and m4 contained also suphate and acid non-sulphate glycans, m3 lacked suphate glycoproteins. Lectin histochemistry showed that mantle mucous cells (m1, m2) expressed GlcNAc and lactosamine terminating glycans. m2 also contained GalNAc terminal or penultimate to sialic acid. m3 was distinguished by mannosylated glycans terminating with lactosamine and m4 by α2,6 sialoglycans. Glycoproteins terminating with lactosamine, Galß1,3GalNAc, and α1,6-linked fucose were a common feature of paralarvae surface layer. Morphometry revealed a significant decrease of m1 and m2 abundance during the first month of life, afterwards the reared paralarvae died. Since the glycopattern did not change during the investigated period, the mantle mucous cells abundance could be related to the Octopus vulgaris paralarvae survival.

Glycopattern analysis of acidic secretion in the intestine of the red-eared slender turtle; Trachemys scripta elegans (Testudines: Emydidae).

The secretion of the goblet cells in the intestine of Trachemys scripta elegans was studied in situ by histochemical methods to analyze the diversity of sugar chains, with particular regard to the acidic glycans. Conventional histochemical stains (Periodic acid-Schiff, Alcian Blue pH 2.5, High Iron Diamine) and binding with ten FITC-labelled lectins combined with chemical and enzymatic pre-treatments were used to characterize the oligosaccharidic chains. The intestine can be divided into three regions, i.e. a duodenum, a small intestine and a large intestine. Goblet cells were observed in all the three tracts and presented an acidic secretion. WGA, LFA, PNA and SBA binding was observed only after desulfation. Glycans secreted by the three tracts consist mainly of sulfosialomucins with 1,2-linked fucose, mannosylated, glucosaminylated and subterminal galactosyl/galactosaminylated residuals. Differences among tracts are quantitative rather than qualitative, with sulfated, galactosaminylated and glycosaminylated residuals increasing from duodenum to large intestine, and galactosylated and fucosylated residuals showing an opposite trend. Variation is observed also between apices and bases of villi in both duodenum and small intestine, where sulphation decreases from the base to the apex and glycosylation shows an opposite trend. Functional implication of these findings is discussed in a comparative context.

A histochemical approach to glycan diversity in the urothelium of pig urinary bladder.

Intracellular glycans in the urothelium of urinary bladder of 10 adult male Landrace pigs were characterized in situ by immunohistochemical detection of Muc1 mucin by anti MUC1 from rabbit, conventional histochemical techniques (Periodic-Acid Schiff, Alcian Blue pH 2.5, High-Iron Diamine), and binding with 13 lectins (PNA, DBA, RCA-I, WGA, SBA, BSI-B4, ConA, AAA, UEA-I, LTA, LFA, MAA-II, SNA) combined with chemical and enzymatic pre-treatments (ß-elimination, desulfation and neuraminidase) to gather reference data for this model animal. Muc1 mucin was detected in the secreting granules of superficial cells and the underlying layer of intermediate cells. The secreting granules in both intermediate cells and superficial cells were rich in carbohydrates, with the oligosaccharidic chains mostly O-linked to proteins. Glycoproteins were prevailing over glycosaminoglycans (GAGs). In both superficial and intermediate cells sulfated and/or sialylated glycans were present, sulfation decreasing in the deeper layers. Lectin-binding detected presence of terminal sialic acid linked mostly in α2,6 to GalNAc, Gal terminal or subterminal to sulfates, GalNAc, GlcNAc, and Fuc, mostly linked in α1,6, α1,3 α1,4 and α1,2 to GlcNAc or Gal, but not to lactosamine chains. Except for fucosylation, the oligosaccharidic chains in the glycoproteins of the urothelium of pig urinary bladder were similar to those linked to human MUC1, which is fundamental in cell adhesion and immunological processes in the urothelium. The co-distribution of Muc1 and saccharidic residues suggests that many of them are linked to the glycoprotein.

Comparative glycopattern analysis of mucins in the Brunner's glands of the guinea-pig and the house mouse (Rodentia).

The mucins secreted by the Brunner's glands and the duodenal goblet cells of the Guinea-pig and the house mouse were compared by conventional and FITC-conjugated lectin histochemistry. Methylation/saponification and sialidase digestion were performed prior to lectin binding to detect the residues subterminal to sulfated groups and sialic acid, respectively. In the Guinea-pig the Brunner's glands produce class-III stable sulfosialomucins. Sialic acid is mostly 2,6-linked to galactose or to N-acetylgalactosamine and is in part O-acetylated in C7, C8, and C9. Sulfated groups are probably linked to sialic acid and N-acetylgalactosamine. Terminal residuals of N-acetylglucosamine, galactose, N-acetylgalactosamine and fucose linked in α1,2, α1,3, and α1,4 are also present. Duodenal goblet cells of the Guinea-pig present a lower number of residuals in respect to the Brunner's glandular ones, with sialic acid and N-acetylgalactosamine subterminal to sulfated groups. In the house mouse the Brunner's glands produce class-III stable neutral mucins, binding to same lectins as in the Guinea-pig except for those specific to sialic acid. A diversity of fucosylated residuals higher than in the Guinea-pig is observed. The mouse duodenal goblet cells lack stable class-III mucins, have little sialic acid and present a lower number of residuals in respect to the correspondent Brunner's glands. Regulation of the acidic intestinal microenvironment, prevention of pathologies and hosting of microflora can explain the observed results and the differences observed between the two rodents.

Histochemical and structural characterization of egg extra-cellular matrix in bufonid toads, Bufo bufo and Bufotes balearicus: molecular diversity versus morphological uniformity.

The extra-cellular matrix of fertilized eggs in the bufonid toads Bufo bufo and Bufotes balearicus was studied to clear the relationships between structural and molecular diversity. Histochemical (PAS, AB pH 2.5 and pH 1.0, Beta-elimination PAS) and lectin-histochemical (Con A, WGA, Succinyl-WGA, PNA, RCA-1, DBA, SBA, AAA, UEA-I, LTA) techniques were used and the observations were made under light and electron microscopy. Both species present a fertilization envelope (FE) and two jelly layers (J1 and J2). The fibers of J2 are shared among the eggs of a clutch in a jelly ribbon. The FE of both species presents neutral glycoproteins, mostly N-linked. In B. bufo there are also residuals of mannose and/or glucose and N-acetylglucosamine. In the FE fibers run parallel to egg's surface or are in bundles or looser hanks with no clear orientation. The J1 layer of both species presents sialosulfoglycoproteins, mostly O-linked, with lactosaminylated, galactosaminylated, glycosaminylated, and fucosylated residuals. A lower amount of galactosaminylated residuals is observed in B. balearicus in respect to B. bufo, whereas the opposite is seen in the amount of fucosylated residuals. The J2 layer is similar in composition to J1 but in B. balearicus there are no glucosaminylated residuals. J layers present fibers and granules that reduce towards J2 . Several microorganisms, in particular blue algae, are observed in the J2 layer of both species. In respect to other species, B. bufo and B. balearicus have a lower number of jelly layers, but a comparable number of glycan types.

Seasonal variation in glycoconjugates of the pedal glandular system of the rayed Mediterranean limpet, Patella caerulea (Gastropoda: Patellidae).

Glycoconjugates secreted by the pedal system of the rayed limpet, Patella caerulea, were characterised in situ by histochemical and lectin-histochemical methods in individuals collected around the annual cycle, in November, March, and June. Stainings with periodic acid-Schiff (PAS), Alcian blue pH 2.5 (AB pH 2.5), Alcian blue pH 1.0 (AB pH 1.0), high-iron diamine-Alcian blue pH 2.5 and lectin binding assays with 9 lectins (Con A, WGA, succinylated-WGA, PNA, DBA, SBA, AAA, UEA-I, LTA) were performed. Four secreting cell types were observed in the sole, one in the peripheric region, and two in the sidewall. Glycoconjugate composition varied among cell types and also in one and the same cell type throughout the year. ß-Elimination followed by PAS and AB pH 2.5 stainings indicated that most saccharidic chains were O-linked to the protein backbone. Secretion by sole and peripheric region was acidic, carboxylated and/or sulfated, whereas that of the sidewall was neutral. Glucosaminylated and 1,4-fucosylated residuals were predominant in the cell types along the year, 1,2-fucosylated residuals being observed only in the sidewall cells in June. Mannosylated and/or glycosylated residuals were observed in all cells mostly in November. Galactosylated/galactosaminylated residuals were present mostly in the sidewall cells and in the sole subepidermal mucocytes in June. Mannosylated and/or glycosylated residuals in November are probably linked to gonad maturation or to higher locomotion and foraging activity, whereas galactosaminylation in the sole cells and 1,2-fucosylation and glucosaminylation in the sidewall cells in June are linked to a prolonged stationary state, increasing water adsorption to counteract dehydration and/or to modulate microbial interactions.

In situ characterization of O-linked glycans of Muc2 in mouse colon.

The characterization of mucus O-linked glycans in the proximal and distal mouse colon was performed by conventional histochemical methods and by lectin histochemistry in combination with enzymatic treatment (PNGase, α1,2 fucosidase, sialidase digestion), with and without prior desulfation. We demonstrated the presence of sialo- and sulfomucins in both the proximal and distal colon of the mouse. In the distal colon the sulfomucins were clearly prevalent, although there were always sialomucins with sialyl residues linked α2,6 to the subterminal galactose. Sialic acid was poorly O-acetylated, especially in the distal colon. The lectin binding pattern indicates a massive presence of fucose α1,2 linked to galactose in O-glycans and smaller quantities of fucose linked α1,6 to N-acetylglucosamine in the core of N-linked glycans. Lectin histochemistry also demonstrated the presence of glycosidic residues of N-acetylglucosamine, N-acetylgalactosamine, and galactose in oligosaccharide chains of highly sulfated mucins.

Diabetic placenta: ultrastructure and morphometry of the term villi.

OBJECTIVE: To verify the ultrafine conformation of term villi in diabetic and normal placentae. Villar dysmaturity and chorangiosis are considered the most frequent findings in diabetic placentae, but their histogenesis is still unclear. STUDY DESIGN: We performed a morphometric study of 38 term villi in 5 diabetic placentae and of 37 term villi of 5 normal placentae in order to know the different extension of endothelial surface (VL), the maximum (D max) and minimum (D min) distance of the vessels from the basal membrane, as well as the exact thickness of basal membrane (MT BM). The villi were examined with transmission electron microscopy, and parameters were automatically acquired with the iTEM software (Soft Imaging System, Münster, Germany). RESULTS: VL results were statistically higher in diabetic placentae than in normal ones. Also D max and D min were higher in diabetic disease. MT BM was not different in the two groups. CONCLUSION: Our findings show that, in the presence of chorangiosis, the vessel surface in diabetic placentae is higher than in normal group, but the vessels are randomly distributed in term villi. The basal membrane is not different in the two groups. Morphometric evaluation seems to be more accurate using ultrafine samples.

Glycopattern analysis and structure of the egg extra-cellular matrix in the Apennine yellow-bellied toad, Bombina pachypus (Anura: Bombinatoridae).

We studied the glycopatterns and ultrastructure of the extra-cellular matrix (ECM) of the egg of the Apennine yellow-bellied toad Bombina pachypus, by light and electron microscopy in order to determine structure, chemical composition and function. Histochemical techniques in light microscopy included PAS and Alcian Blue pH 2.5 and 1.0, performed also after ß-elimination. Lectin-binding was tested with nine lectins (AAA, ConA, DBA, HPA, LTA, PNA, SBA, UEA-I, WGA). An inner fertilization envelope (FE) and five jelly layers (J1-J5) were observed, differing in histochemical staining, lectin binding and ultrastructure. Most glycans were O-linked, with many glucosamylated and fucosylated residues. The fertilization envelope presented a perivitelline space and a fertilization layer, with mostly neutral glycans. The jelly layers consisted of fibers and granules, whose number and orientation differed between layers. Fibers were densely packed in J(1) and J(4) layers, whereas a looser arrangement was observed in the other layers. Jelly-layer glycans were mostly acidic and particularly abundant in the J(1) and J(4) layers. In the J(1), J(2) and J(5) layers, neutral, N-linked glycans were also observed. Mannosylated and/or glucosylated as well as galactosyl/galactosaminylated residues were more abundant in the outer layers. Many microorganisms were observed in the J(5) layer. We believe that, apart from their functions in the fertilization process, acidic and fucosylated glycans could act as a barrier against pathogen penetration.

Co-distribution of glycoconjugates and H(+), K(+)-ATPase in the parietal cells of the greater horseshoe bat, Rhinolophus ferrumequinum (Schreber, 1774).

Histochemical, lectin-histochemical, and immunohistochemical analyses were performed on parietal cells of the greater horseshoe bat, Rhinolophus ferrumequinum, to clarify the composition and distribution of oligosaccharide chains in the beta-subunit of the protonic pump H(+),K(+)-ATPase. PAS, Alcian Blue (pH 2.5) and Alcian Blue (pH 1.0) stainings detected only neutral glycoconjugates. Lectin-binding analyses included LTA, UEA-I, ConA, SBA, BSI-B4, AAA, DBA, PNA, and WGA. WGA-and PNA-bindings were also tested after beta-elimination to detect O-linked glycans. Parietal cells were negative for binding to LTA and UEA-I, and to PNA and WGA after beta-elimination, indicating the lack of (1,2) fucosylated residues and of N-linked glycans, respectively. Immunohistochemical tests with anti-alpha- and anti-beta-H(+),K(+)-ATPase were positive. Two alternative patterns of glycoconjugate distribution were found, i.e. a perinuclear and a diffuse one, indicating localization in the intracellular canaliculus and in the tubulovesicular system of the parietal cells, respectively. Both the subunits of the H(+),K(+)-ATPase and the galactosyl/galactosaminyl residues were co-distributed in both the perinuclear and the diffuse patterns, suggesting that the residues are part of the protonic pump. Glycosyl/glycosaminyl and mannosyl groups were concentrated in the tubulovesicular system, and fucosylated residues were found almost exclusively in the intracellular canaliculi; thus they are probably not included in the oligosaccharide chains of beta-H(+),K(+)-ATPase. These findings indicate that the oligosaccharide chains linked to the beta-H(+),K(+)-ATPase subunit in R. ferrumequinum have distinct features compared to the other mammals studied and confirms the taxon specificity of the chains in the proton pump.

Altered membrane glycoprotein targeting in cholestatic hepatocytes.

BACKGROUND: Hepatocytes are polarized epithelial cells with three morphologically and functionally distinct membrane surfaces: the sinusoidal, lateral and canalicular surface domains. These domains differ from each other in the expression of integral proteins, which concur to their polarized functions. We hypothesize that the cholestasis-induced alterations led to partial loss of hepatocyte polarity. An altered expression of membrane proteins may be indicative of functional disorders. Alkaline liver phosphatase (ALP), one of the most representative plasma membrane glycoproteins in hepatocytes, is expressed at the apical (canalicular) pole of the cell. Because the release of ALP protein in the bloodstream is significantly increased in cholestasis, the enzymatic levels of plasma ALP have major relevance in the diagnosis of cholestatic diseases. Here we assess the cholestasis-induced redistribution of membrane glycoproteins to investigate the ALP release. MATERIALS AND METHODS: We performed enzymatic histochemistry, immunohistochemistry, lectin histochemistry, immunogold and lectin-and immunoblotting studies. Experimental cholestasis was induced in rats by ligation of common bile duct (BDL). RESULTS: The BDL led to altered membrane sialoglycoprotein targeting as well as to ultrastructural and functional disorders. Disarrangement of the microtubular system, thickening of the microfilamentous pericanalicular ectoplasm and disturbance of the vectorial trafficking of membrane glycoprotein containing vesicles were found. CONCLUSIONS: Altogether, results indicate that the cholestasis-induced partial loss of hepatocyte cell polarity leads to mistranslocation of ALP to the sinusoidal plasma membrane from where the enzyme is then massively released into the bloodstream.