Sialoglycoderivatives of bovine submandibular gland identified in situ by histochemical techniques combined with lectins.

We employed sialidase procedures followed by lectin stainings combined with oxidizing and deacetylating agents to visualize the distribution and sequentiate sialoglycoconjugates in the bovine submandibular gland. In particular we evidenced in acinar and ductal cells the dishomogeneous presence of sialic acids acetylated in the polyhydroxy side chain (C7, C8, C9), whereas O-acetyl substituents at position C1 and/or C4 were not found. Sialoglycoderivatives were also differentiated by the occurrence of penultimate sugars; indeed the dimers sialic acid-(alpha 2----3,6)-beta-galactose and sialic acid-(alpha 2----6)-alpha-N-acetylgalactosamine were identified. Using such technique we supported further the possibility to develop methods for the identification of the positions of O-acetyl groups and the reconstruction of terminal disaccharides within surface and cytoplasm glycoconjugates. in situ the distribution of different O-acylated sialoglyco-derivatives in the bovine submandibular gland. To this purpose we employed oxydizing and deacetylating agents combined with sialidase digestion and lectin binding.     

Determination in situ of neutral and acidic fucose-containing oligosaccharides in the bovine submandibular gland

Lectins conjugated to horseradish peroxidase in combination with selected exoglycosidase digestion procedures were used to localize fucoglycoconjugates in the bovine submandibular gland. In particular, sequential treatments were employed to determine the distribution of neutral and acidic fucose-containing oligosaccharides that were previously shown to be present by biochemical techniques. Information was obtained on the distribution of the acidic oligosaccharide A-1a, -Fuc(12)--Gal-(14)--GlcNAc-(13)-[-NeuAc-(26)]-GalNAc-ol, which was sequenced in situ and localized in acinar cells.     

Classification of bovine muscle fibres by different histochemical techniques

The classification of bovine muscle fibres is of particular interest for the food industry because meat tenderness depends in part on the proportion of the different types of fibres. It is, therefore, important to define reliable methods for classifying fibre types. There are several classification systems. One is based on contractile type alone, as revealed by myofibrillar ATPase activity or with antibodies against myosin heavy chain. Others take both contractile and metabolic types into account. In this study, the classifications of fibres obtained by these three systems were compared on the semitendinosus and longissimus thoracis muscles of 35 Charolais bulls. Only the use of antibodies allowed the identification of a proportion of hybrid fibres containing two isoforms of fast myosin heavy chain (2a and 2b). In addition, the combination of metabolic types showed that the metabolism of these hybrid fibres differed according to the muscle.     

Identification of peroxidase-positive astrocytes by combined histochemical and immunolabeling techniques in situ and in cell culture.

A subpopulation of astrocytes in the vertebrate brain and in cysteamine-treated brain cell cultures contain cytoplasmic granules which exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic endogenous peroxidase activity. Visualization of these cells at the light microscopic level is confounded by the nonspecificity of the various histochemical methods routinely employed. In an attempt to circumvent this problem, we assayed for peroxidase-positive astrocytes using various combinations of diaminobenzidine (DAB) histochemistry and immunolabeling for the astrocyte-specific marker glial fibrillary acidic protein (GFAP). We determined that (a) DAB histochemistry in conjunction with avidin-biotin-immunoperoxidase labeling for GFAP specifically detects peroxidase-positive astrocytes in situ and (b) DAB histochemistry combined with indirect immunofluorescence for GFAP effectively demonstrates these cells in cysteamine-treated brain cell cultures.     

Glycosidic residues in the mouse submandibular gland: biochemical determination and visualization with plant lectins

Biochemical and histochemical studies were carried out on the mouse submandibular gland to define the presence and the localization of glycosidic residues. The results obtained by applying peanut, winged pea, wheat germ, soybean and Con A lectins to unfixed and fixed sections tallied completely with biochemical determinations of the various glycosidic residues. The data from this study have been compared with those obtained by other authors who had performed biochemical and histochemical studies on the gland; similarities and discrepancies as to the presence and the localization of the sugars are discussed.     

Morphological and histochemical study of the submandibular gland in Praomys (Mastomys) natalensis

The submandibular gland in female and male Praomys (Mastomys) natalensis (a South African multimammate rodent) was studied using light microscopy and techniques for the demonstration of carbohydrates. Hematoxylin and eosin stain revealed the presence of a single secreting component that gave a strongly positive PAS reaction. Limiting elements of the granular tubules gave a weakly positive PAS reaction. Acidic glycoproteins were evidenced only in granules of the acinar component.     

Subcellular site of the biosynthesis ofO-acetylated sialic acids in bovine submandibular gland

Bovine submandibular glands were homogenized and fractionated under conditions which yielded subcellular fragments from mainly one cell type, the mucous acinar cell, as judged by morphological analysis of the glands before and after homogenization. The majorN-acetylneuraminate-9(7)-O-acetyltransferase activity was detected in the cytosolic fraction, a result supported by the high specific radioactivity of free sialic acids isolated after [¹⁴C]acetate-labelling experiments. Separation of membranes on a Ficoll density gradient gave six fractions which were analyzed biochemically and morphologically. The particulate activities of acetyltransferase and sialyltransferase were found in fractions containing smooth and mitochondrial membranes. MembraneO-acetyl sialic acids were present at the highest levels in these fractions and also had the highest specific radioactivity after [¹⁴C]acetate-labelling experiments. Significant amounts of theO-acetyltransferase activity also occur in the cytosol and are consistent with a model ofO-acetyl sialic acid biosynthesis involving both cytosolic and smooth membrane sites ofO-acetylation.     

Arginine vasotocin mRNA revealed by in situ hybridization in bovine pineal gland cells

Arginine vasopressin (AVP) is the main antidiuretic hormone in mammals and arginine vasotocin (AVT) in submammalian vertebrates. The possibility that the genetic material encoding AVT is maintained in mammals is controversial. In this study, we investigated by radioactive in situ hybridization the possible presence of the mRNA encoding AVP and AVT, and using immunocytochemistry the presence of structures immunoreactive for AVP and AVT in the bovine pineal gland. In situ hybridization was performed by use of ³⁵S-labelled oligoprobes. Immunocytochemistry was performed using specific polyclonal rabbit antibodies and the avidin-biotin-complex method. In situ hybridization revealed positive signals for both AVP mRNA and AVT mRNA in a few cells scattered throughout the pineal body. Immunocytochemistry revealed thin AVP-immunoreactive fibres in the pineal stalk and the pineal gland. It also revealed staining of several AVT-immunoreactive nerve fibres in both the pineal stalk and the gland. In addition, polyhedral, neuron-like cell bodies from which two to three processes emerged were also AVT-immunoreactive. Thus, our investigation shows the presence of AVP/AVT-immunoreactive cellular structures in the bovine pineal gland. Our data further show the presence of mRNAs encoding both AVT and AVP. We therefore suggest that AVT mRNA is translated into an AVT-like peptide in the bovine pineal.     

Histochemical observations on endogenous lactoperoxidase activity of the bovine submandibular salivary gland

Synopsis Seromucous demilunar cells of glutaraldehyde-fixed bovine submandibular salivary glands are intensely stained when sections are incubated in a benzidine-or a 3,3-diaminobenzidine-hydrogen peroxide medium in the pH range 6.0–9.0 whereas mucous acinar cells are completely unreactive. The histochemical reaction is completely inhibited by 3-amino-1,2,4-triazole. In contrast 2,4-dichlorophenol or potassium cyanide has little or no effect on the staining of demilunar cells. Striated duct cells also display a positive reaction with the diaminobenzidine method; this staining reaction, however, is most intense at pH 6.0. Furthermore, this reaction is markedly affected by potassium cyanide. The positive histochemical benzidine and diaminobenzidine reactions of demilunar cells probably corresponds to endogenous lactoperoxidase activity. On the other hand, the positive reaction shown by striated ducts, with optimal staining at pH 6.0 and which is completely inhibited by potassium cyanide, seems to be due to cytochromal oxidation of diaminobenzidine.     

Characterization of the major and minor mucus glycoproteins from bovine submandibular gland

Two mucins were isolated from bovine submandibular glands and termed major and minor on a quantitative basis. The major mucin representing over 80% of the total glycoprotein fraction contained 37% of its dry weight as protein in contrast to 62% for the minor mucin. Differences in the amino acid composition reflected the higher proportion of typically non-glycosylated peptide in the minor mucin. The molar ratio ofN-acetylgalactosamine to serine plus threonine was 0.82 in major and 0.65 in minor mucins, indicating a lower degree of substitution of potential glycosylation sites in the minor mucin.Differences in the carbohydrate composition were found largely related to the sialic acids, with higher relative amounts ofN-glycoloylneuraminic acid in the minor mucin. In addition, the proportion of di-O-acetylated sialic acids was higher in the major mucin. The rate of sialidase action on the two mucins could be correlated with the content ofN-glycoloylneuraminic acid in each glycoprotein. There was no difference in the type of oligosaccharide found in each mucin and the differences in relative proportions reflected the monosaccharide composition for the two mucins. Gel filtration on Sepharose CL 2B showed a lower molecular weight distribution for the minor in contrast to the major mucin which was partially excluded. Density gradient centrifugation reflected this variation. SDS-PAGE demonstrated a regular banding pattern for the major mucin with a lowest subunit size of 1.8×10⁵ Da and aggregates in excess of 10⁶ Da, while the minor mucin ranged from 3.0 × 10⁵ to 10⁶ Da. The chemical composition of the isolated mucins was compared with previous histochemical analysis of mucin distribution in bovine submandibular glands and indicates a possible cellular location for each mucin.Abbreviations PBS 0.01m sodium phosphate buffer, pH 7.3, containing 0.15m NaCl - Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycoloylneuraminic acid - GalNAc-ol N-acetylgalactosaminitol     

Immunohistochemistry combined with periodic acid-Schiff for bovine mammary gland with protothecal mastitis

Immunohistochemistry (IHC) of bovine cytokeratin combined with periodic acid-Schiff (PAS) was applied to study the pathogenesis, localization and distribution of Prototheca zopfii in bovine mammary protothecosis. The standard immunohistochemical procedure using anti-bovine cytokeratin was employed before and after PAS staining to optimize this combined method. The best results were obtained when IHC procedures were performed first. Most of the epithelial cells reacted strongly with the pancytokeratin antibody. Protothecal cell walls stained well with PAS. Algal organisms were present within the lumen and between the epithelial lining and basement membrane of the affected alveoli, but not inside the positive mammary epithelial cells. This combined staining method resulted in clear alveolar epithelial detail and good contrast between the epithelial cells and algae, and contributed to studying the pathogenesis of P. zopfii in mammary protothecosis.     

Immunohistochemistry combined with periodic acid-Schiff for bovine mammary gland with protothecal mastitis

Immunohistochemistry (IHC) of bovine cytokeratin combined with periodic acid-Schiff (PAS) was applied to study the pathogenesis, localization and distribution of Prototheca zopfii in bovine mammary protothecosis. The standard immunohistochemical procedure using anti-bovine cytokeratin was employed before and after PAS staining to optimize this combined method. The best results were obtained when IHC procedures were performed first. Most of the epithelial cells reacted strongly with the pancytokeratin antibody. Protothecal cell walls stained well with PAS. Algal organisms were present within the lumen and between the epithelial lining and basement membrane of the affected alveoli, but not inside the positive mammary epithelial cells. This combined staining method resulted in clear alveolar epithelial detail and good contrast between the epithelial cells and algae, and contributed to studying the pathogenesis of P. zopfii in mammary protothecosis.     

Endogenous lectins of bovine pancreas

Affinity chromatography of salt and detergent extracts from bovine pancreas on glycosylated or glycoprotein-linked Sepharose 4B resulted in purification of different carbohydrate-binding proteins. Three species of proteins with molecular masses of 16 kDa, 35 kDa and 64 kDa exhibiting specificity for beta-galactosides, but none with preferential specificity for alpha-galactosides, were isolated from salt and detergent extracts. No Ca2+ was required for binding. Mannan-binding proteins of 37 kDa, 47 kDa and 94 kDa without Ca2+-requirement were only found in the salt extract. No other mannan-binding activity could be detected. Fucose-binding proteins of 34 kDa, 62 kDa and 70 kDa exhibiting Ca2+-requirement for binding were present in the salt extract and two proteins with 62 kDa and 70 kDa in detergent extract. The different fractions showed agglutination activity when assayed with rabbit erythrocytes. Thus they can be defined as lectins.     

In situ localization of mRNA for epidermal growth factor in the submandibular gland of the mouse

Epidermal growth factor (EGF) is a polypeptide originally isolated from the mouse submandibular gland, where it is localized immunocytochemically in cells of the granular convoluted tubules (GCT). cDNAs encoding the precursor of mouse submandibular EGF have been cloned (Scott et al. Science 221:236, 1983; Gray et al. Nature 303:722, 1983). A fragment of one of these clones, pmegf10, containing the EGF coding region, was tritium-labeled by nick-translation and used as a probe for in situ hybridization to EGF mRNA. A specific hybridization signal for EGF mRNA was seen only in mature or developing GCT cells. The intensity of the signal was stronger in glands of intact males than in females or in castrated males. In glands of castrates treated with testosterone, or of intact females treated with triiodothyronine (T3), the signal was comparable to that in intact males. In glands of males treated with T3 the intensity of the signal was stronger than in untreated males. A weak to moderate signal was seen in developing GCT cells of 20-day-old males but not females. Hybridization for 3 days gave a stronger signal than that for 1 day. No signal was seen in either sex at 10 days of age, or in control preparations exposed to labeled DNA of pBR322. The presence of EGF mRNA exclusively in GCT cells provides strong evidence that these cells are the only site of synthesis of EGF in the submandibular gland. In situ hybridization with this cDNA probe will provide a sensitive method to determine possible cellular sites of EGF production outside of the submandibular gland.     

Excision of the submandibular gland by an intraoral approach

To improve the outcome in patients with benign diseases of the submandibular gland, we have developed an entirely intraoral technique for excision of the submandibular gland. This procedure is anatomically safe and can be performed with minimal morbidity. We believe the essential surgical steps are as follows: (1) infiltration with Xylocaine plus epinephrine with an adequate waiting period for hemostasis; (2) careful identification of the submandibular duct/lingual nerve relationship; (3) anterior retraction of the mylohyoid muscle to expose the superficial lobe; (4) superiorly directed, extraoral, manipulation of the submandibular gland; and (5) close and blunt dissection to the gland laterally to avoid injury to the facial artery and vein.     

Use of Phaseolus vulgaris leukoagglutinating lectin in histochemical and blotting techniques: a comparison of digoxigenin- and biotin-labelled lectins

An increase in the number of 1,6 branches of the trimannosyl core of asparagine-linked oligosaccharides has been shown to be directly correlated with the metastatic potential of cultured tumour cells. The Phaseolus vulgaris leukoagglutinating lectin (PHA-L) binds to 1,6 branches of tri- and tetra-antennary oligosaccharides. We have applied digoxigenin- and biotin-conjugated PHA-L to establish a non-radioactive detection system for 1,6 branches, which can be used in lectin blotting as well as light and electron microscopic cytochemistry. For this purpose the HCT116 human colon carcinoma cell line and colon carcinoma tissue were investigated. Digoxigenin-conjugated PHA-L in conjunction with alkaline phosphatase-conjugated anti-digoxigenin antibodies was superior to biotin-conjugated PHA-L in lectin blotting with respect to sensitivity and specificity. Similarly, the digoxigenin conjugated PHA-L in conjunction with gold-labelled anti-digoxigenin antibodies resulted in more intense specific staining and lower background compared to biotin-conjugated PHA-L visualized with a streptavidin immunogold complex. The specificity of lectin binding in blotting and cytochemical studies was demonstrated by the absence of staining when the lectin was omitted or preabsorbed with glycoprotein, and following pretreatment of the cellular homogenates or tissue sections by N-glycosidase F. Our results demonstrate that digoxigenin-conjugated PHA-L provides high sensitivity and specificity for histochemical and blotting techniques and is amenable for quantification. The technique should have applications in tumour research.     

N-acetyl-9-O-L-lactylneuraminic acid, a new acylneuraminic acid from bovine submandibular gland.

The acylneuraminic acid fraction, obtained on mild acid hydrolysis of glycoproteins from bovine submandibular glands, contains approximately 2% N-acety-9-O-L-lactylneuraminic acid. The compound has been isolated and purified by ion-exchange and cellulose column chromatography. The structure has been elucidated using thin-layer chromatography, colorimetry, gas-liquid chromatography/mass spectrometry, periodate oxidation and specific lactate dehydrogenases. An evaluation of the different analytical methods is given.