Histochemical demonstration of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues by means of almond glycopeptidase digestion

Almond glycopeptidase is an enzyme which cleaves specifically beta-aspartylglucosylamine linkages in glycoproteins with asialo-carbohydrate moieties. With this enzyme, it was possible to demonstrate the localization of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues. In these tissues, the oligosaccharides were shown to react positively for a series of histochemical procedures for neutral complex carbohydrates such as periodic acid-Schiff (PAS), peroxidase-labelled Ricinus communis agglutinin-I-diaminobenzidine (PO-RCA-DAB) and concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB). The asparagine-linked carbohydrates were localized in the placental villi, blood vessels and perivascular tissues and the umbilical cord blood vessels and matrix. The results of previous biochemical analyses performed upon the same tissues (Takahashi et al., 1981) have corroborated the results of the histochemical studies. The present results appear to substantiate the usefulness of almond glycopeptidase for the histochemical demonstration of the particular oligosaccharides of glycoproteins in tissues in general.     

Effects of digestion with N-oligosaccharide glycopeptidase upon certain lectin-peroxidase-diaminobenzidine reactions of glycoproteins in mammalian and avian tissues

Summary To determine the histochemical localization of asparagine-linked oligosaccharides of glycoproteins in a series of different mammalian and avian tissues, the effects of digestion with N-oligosaccharide glycopeptidase upon certain lectin-peroxidase-diaminobenzidine reactions of the histological structures involved have been studied by light microscopy. Throughout the tissues examined, asparaginelinked oligosaccharides of glycoproteins were localized mainly in histological structures of connective and muscular tissues, but were hardly or not visualized in those of epithelial tissues. these results appear to lead to the concept that connective and muscular tissues represent the main sites where plasma types of glycoproteins are involved in mammalian and avian species.     

Glycoproteins responsive to the neural-inducing effect of Concanavalin A in Cynops presumptive ectoderm

To examine the possible occurrence of receptors in the ectodermal cell surface which apparently mediates the neural-inducing stimulus, a further experiment by using Con A was done in combination with the enzyme treatments. The presumptive ectoderm explants of Cynops gastrula were first treated with neuraminidase to remove sialic acid. Prior to the Con A treatment, the explants were treated with almond glycopeptidase, which cleaves the asparagine linkage between protein and oligosaccharide in glycoprotein and releases the oligosaccharide moiety intact containing mannose residue from the substrate. No neural induction occurred. When the explants were not treated with almond glycopeptidase, the neural induction frequency was found to be the same as that of the explants treated with only Con A. Biochemical analyses showed that when the fixed ectoderm explants were treated with almond glycopeptidase, several oligosaccharides were released and then fractionated by means of Bio-Gel P-4 filtration. Based on the strict specificity of almond glycopeptidase, these oligosaccharides are unmistakably asparagine-linked oligasaccharides with mannose residues. We discuss the hypothesis of involvement of glycoproteins in the first step of molecular events in the neural induction mechanism.     

Analysis of asparagine-linked oligosaccharides from plasma membranes of rat normal liver and ascites hepatoma cells

Isolated plasma membranes from rat liver and ascites hepatoma cells were shown by SDS-polyacrylamide gel electrophoresis and concanavalin A reactivity to contain a variety of glycoproteins having asparagine-linked oligosaccharides. Membrane oligosaccharides were released by almond glycopeptidase digestion, and the pyridylamino derivatives were analyzed by high-performance liquid chromatography. Forty-four percent of the total carbohydrates in the original membranes were released and suggested to be of the complex type. Hepatoma membranes showed different oligosaccharide patterns from normal.     

Changes in the Microvasculature and Interstitium of Aged Human Mandibles and Maxillae1

The present study describes the age changes to the microvasculature and connective tissue interstitium of the osteons and periosteums of aged human mandibles and maxillae. The mandibles and maxillae obtained from 14 and 19 year old males, respectively, were also studied. In the nutrient canals of the aged osteons, the walls of the arterioles and venules stained intensely PAS positive, and alcian blue negative. The walls of the blood capillaries were thick and strongly PAS positive. There was a deposition of PAS positive material in the connective tissue stroma of the nutrient canals which progressed to the obliteration of the canal space. Many of the nutrient canals exhibited diffuse calcification within the connective tissue interstitium localized around the blood vessels. The lacunae and canaliculi of those osteons in which the nutrient canals were partially or completely obliterated were filled with PAS material. None of these histochemical changes were seen in the osteons of young individuals. The microvasculature of the aged periosteum showed similar changes. The periosteal tissue consisted of thick collagenous bundles and few osteogenic cells. There was a thin darkly stained amorphous calcified layer forming the bone surface.     

The periodic acid-schiff reaction in the adrenal medulla

Summary The PAS reaction in the adrenal medulla of rat, rabbit, hamster, ox, pig and sheep was investigated. The medullary cells were positive in cryostat sections and potassium dichromate fixed material but not in formaldehyde fixed paraffin sections. The latter result is due mostly to the extraction of PAS positive lipids and loss of PAS positive proteins. No glycogen was detected in the chromaffin cells histochemically. The catechol amines played no part in the PAS reaction unless the fixative contained dichromate. The connective tissue elements were also PAS positive, and the nerve fibres in ox, sheep and pig. Periodate cannot be used to differentiate between adrenaline and noradrenaline storing cells.     

N-glycosylation of purified rat and rabbit hepatic UDP-glucuronosyltransferases

Five UDP-glucuronosyltransferases (UDPGTs) have been isolated to apparent homogeneity from rat and rabbit liver and have been characterized for their glycoprotein nature by reacting these proteins with commercially available endo- and exoglycosidases. The enzymes studied were rat hepatic p-nitrophenol, 17 beta-hydroxysteroid, and 3 alpha-hydroxysteroid UDPGTs and rabbit hepatic p-nitrophenol and estrone UDPGTs. Hydrolysis of oligosaccharide moieties was evidenced by an increase in the mobility (decreased apparent molecular weight) of the protein subunits after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified rabbit hepatic estrone and p-nitrophenol UDPGTs were hydrolyzed by almond glycopeptidase A and endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus (endo H), but not by endo-beta-N-acetylglucosaminidase D from Diplococus pneumoniae (endo D) suggesting that these transferases are glycoproteins of the high mannose type and not of the complex type. Likewise, purified rat hepatic 3 alpha-hydroxysteroid and p-nitrophenol UDPGTs were substrates for glycopeptidase A and endo H but not for endo D. One enzyme, 17 beta-hydroxysteroid UDPGT, was not glycosylated since it was not hydrolyzed by any of the three endoglycosidases. All four glycosylated UDPGTs could serve as substrates for jack bean alpha-mannosidase, confirming the high mannose nature of the oligosaccharide. Deglycosylation of the purified UDPGTs by endo H did not have an effect on the catalytic activities of these proteins.     

Electron microscopic autoradiography of 35SO4-labelled material closely associated with collagen fibrils in mammalian synovium and ear cartilage.

Synopsis Electron microscopic autoradiography of connective tissue obtained from mice and rabbits previously injected with³⁵SO₄ indicated that sulphated proteoglycans are localized on collagen fibrils. Ruthenium Red-positive transverse belts surrounding fibrils near the a-bands were heavily labelled, but fine lateral filaments of Ruthenium Red-positive material were not. These filaments, which interconnect collagen fibrils in a variety of connective tissues may represent linear aggregations of hyaluronic acid, glycoproteins and non-sulphated or long-lived sulphated proteoglycans.     

Origin of periodic acid-Schiff-reactive glycoprotein in human eccrine sweat

To evaluate the possible involvement of ductal blockade with periodic acid-Schiff (PAS)-positive materials in the mechanism of hidromeiosis in humans, skin slices were incubated with methacholine for 2 h and PAS-positive materials localized histologically in the ductal lumen. In 20% of the glands complete ductal blockade with PAS-positive materials was noted. The characteristics and origin of such PAS-positive glycoproteins in human sweat were then studied using various electrophoretic techniques. One-dimensional sodium dodecylsulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) demonstrated considerable individual variation in the electrophoretic pattern; however, four major bands at 45, 28, 20, and 18K shared by different individuals, were PAS positive. Further studies using two-dimensional SDS-PAGE, immunodiffusion and immunoaffinity chromatography demonstrated that the PAS-positive glycoproteins are not derived directly from serum because they are electrophoretically and antigenically distinct from serum proteins, including alpha 1-glycoprotein, alpha 2-HS-glycoprotein, and alpha 1-antitrypsin. Since only dark cell granules are densely stained in the histochemical PAS staining, and because antiserum produced against the PAS-positive band selectively stained cells facing the secretory coil lumen (which are most likely dark cells), it is suggested that PAS-positive sweat glycoproteins are derived predominantly from the dark cells.     

The core molecule from type H proteoglycan. Release of mannose-containing oligosaccharides by digestion with N-oligosaccharide glycopeptidase.

Chick-embryo cartilage contains a unique set of proteoglycans. Type H proteoglycan (PG-H) is the most abundant, constituting over 90% of the total cartilage hexuronate. We previously showed that treatment of PG-H with chondroitinase ACII and keratanase yields a protein-enriched core molecule [PG(-CS,KS)] with enzymically modified linkage oligosaccharides of the chondroitin sulphate and keratan sulphate chains. We report here that further treatment of PG(-CS,KS) with pepsin and N-oligosaccharide glycopeptidase (almond glycopeptidase) released four distinct types of mannose-containing oligosaccharide. Two of them were shown to be: (Formula: see text). Of the mannose-containing glycopeptides formed by pepsin digestion, about 40% (as mannose) were resistant to N-oligosaccharide glycopeptidase. Since the resistant fraction was enriched in keratan sulphate remnants, it is suggest that the mannose-containing oligosaccharides in this fraction represent those located in a keratan sulphate-enriched region of PG-H.     

Almond glycopeptidase acting on aspartylglycosylamine linkages. Multiplicity and substrate specificity

The glycopeptide preparation that has been isolated from almond emulsin and acts on β-aspartylglycosylamine linkages in glycopeptides was separated into three active fractions by DEAE-cellulose column chromatography. The three discrete species of glycopeptidase (Groups A, B and C) have been purified 30-, 136- and 99-fold, respectively. The optimum pH value of Group A was 6.0 and those of Groups B and C, 5.0. Isoelectric points of Groups A, B and C were pH 7.7, 8.6 and 8.7, respectively. All three glycopeptidases hydrolyzed quantitatively glycopeptides with 3.11 amino acid residues prepared from stem bromelain, ovalbumin and ovotransferrin. Group C preferred glycopeptides with shorter peptide chains, whereas Groups A and B preferred those with longer chains. Glycopeptidase Group A also hydrolyzed intact glycoproteins such as stem bromelain, ovalbumin, Taka-amylase A and desialyted human transferrin.     

Some regularities of the regeneration of the musculature of internal organs]

The paper summarizes the data of the author and his co-workers on regulation of the musculature of vessels, digestive tract, uterus, ureters and urinary bladder. The smooth muscle cells have different degrees of differentiation. The muscular tissue of the vessels is most differentiated. The uterus musculature is very plastical. After injury mature myocytes are the first to undergo destruction. The intermedial substance is more stable. Myoblasts, young elements of the fibroblast row and the subendothelial layer cells are the origin of muscular regeneration. Figures of mitosis and amitosis are noted. Mature myocytes and intercellular substance are formed in the process of differentiation of the regeneration. The content of RNP in the regeneration cells is high, but in the process of differentiation of its elements it becomes lower. The DNP level has inconsiderable fluctuations. In early experiments PAS-positive substances are revealed in greater degree than in later ones. The content of acid mucopolysaccharides decreases in the process of fibrillogenesis. In all internal organs under study the muscular tissue regenerated. The degree of differentiation, severity of the lesion and functional peculiarities of the organ determine the completeness of the tissue reparation. The musculature of the intestinal tract and vessels regenerates more completely. Mighty layers of connective tissue with de novo formed blood vessels are disposed among the bundles of the repaired muscular tissue of the uterus and urine bladder wall. Simultaneously a part of regeneration cells are destroyed. These are two sides of a single process of development.     

DNA biosynthesis in the muscular and connective tissue cells of the heart in experimental myocardial infarct

In 60 rabbits with experimental myocardial infarction induces by ligation of the anterior branch of the left coronary artery, DNA synthesis was studied by means of H3-thymidine in muscular and connective tissue cells depending on the period of myocardial infarction. The development of myocardial infarction in the cardiac muscle was stated to be accompanied by activation of DNA synthesis in the connective tissue cells not only in the necrotic zone but in the adjacent, as well as in distant areas of the myocardium and in stromal cells of the auriculum. Indices of H3-thymidine labeled nuclei were of high value during the acute period of myocardial infarction and gradually decreased with the time elapsed since the operation. During the period of the myocardical infarction organization high activity in DNA synthesis was revealed in connective tissue elements of the epicardium and the subepicardial zone of the heart. Myocardial cells of the cardiac auriculi incorporated H3-thymidine but extremely seldom-single labels per thousands of nuclei. In the auriculi with application of prolonged sessions of the labeled precussor introduction, DNA synthesising nuclei were revealed, sometimes with paired nuclei of cardiomyocytes.     

Axonal transport of newly synthesized glycoproteins in a single identified neuron of Aplysia californica

Increasing amounts of glycoprotein synthesized from L-[³H]fucose injected into the cell body of R2, an identified Aplysia neuron, were found in the right pleuro-abdominal connective. Autoradiography revealed that the glycoproteins were localized in the axon of R2. Glycoproteins appearing in the axon presumably were synthesized in the cell body, since no significant incorporation was observed when [³H]fucose was injected directly into the axon. [³H]glycoproteins were detected in the connective after a delay of 1 h after intrasomatic injection. Thereafter, transport from the cell body was rapid, and by 10 h after injection, 45% of the total neuronal [³H]glycoprotein had appeared in the axon. By analysing the radioactivity in cell body and connective 4, 10, and 15 h after injection, we found that [³H]glycoproteins were transported selectively compared to nonmacromolecular material. Sequential sectioning of the connective revealed that [³H]glycoproteins were transported in discrete waves. The population of membrane-associated [³H]glycoproteins in the axon differed from that in the cell body. Two of the five somatic components appeared to be transported preferentially. In addition a new component appeared in the axon 10 h after injection.     

Quantitative estimation of lipid-laden cells in atherosclerotic lesions of the human aorta.

The intima of the adult human aorta consists of three sublayers: a muscular layer lying next to the media, a median hyperplastic layer and an innermost connective tissue layer, adjoining the lumen. The cells inhabiting these sublayers were isolated by the method of alcoholic-alkaline dissociation from grossly normal areas, fatty streaks and atherosclerotic plaques. The populations obtained contained cells with different numbers of cytoplasmic inclusions and a number without any. In unaffected intima and in fatty streaks, the cells with lipid inclusions were found predominantly in the outermost intimal layer including the connective tissue and in part of the median hyperplastic layer. In the superficial layer of unaffected intima and the fatty streak, these cells accounted for 15 and 25% of the total cell population, respectively. In the plaque, most cells with lipid inclusions were localized in the median hyperplastic layer of the intima (10%). The muscular layer was characterized by the lowest content of cells with lipid inclusions both in the unaffected intima and atherosclerotic lesions (from 0.75% in unaffected intima to 5% plaques). Among the intimal smooth muscle cells of various shapes, the cells with lipid inclusions were most often found in the stellate cell subpopulation (5-35%). A possible role of stellate cells in atherogenesis is discussed.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Biomorphosis of the human fallopian tube mucosa; a contribution to the problem of morphology and aging of the uterine tube. II. Histochemical findings

The present histochemical findings of investigations on the biomorphosis of the human tubal mucosa indicate that mucosubstances and glycogen localized in the epithelium exhibit age-dependent changes with regard to occurrence and localization. In the embryo-fetal time PAS-positive, diastase-resistant substances are localized in the epithelium, at first basally, and later perinuclearly. In the neonatal phase the distal tubal epithelium has only a weak PAS reaction, and the proximal epithelium has a detectable supranuclear activity. In the end of the 1st decade of the life the epithelium possesses a periodate reactive diastase-sensitive material densely deposited in the preampullar and ampullar parts of the uterine tube, preferably. Afterwards PAS-positive diastase-sensitive and diastase-resistant substances, respectively, are regularly present, in which in the fertile age of the women a regular pattern of the PAS activity can be demonstrated. In the period of the regressive age it is possible to establish a increasing disturbance of the usual cellular picture of the tubal epithelium. In connection with the structural changes a increase of histochemical different reacting cell groups is evident. As a result, a dissociated cellular picture has developed. Epithelial glycoproteins and glycogen can be detected in the mucosa up to the phase of the senium.     

The pineal gland of the Indian palm squirrel, Funambulus pennanti (Wroughton)

In the adult palm squirrel, F. pennanti the pineal is a club shaped, elongated structure with a connective tissue capsule. It consists of various types of pinealocytes, glial cells, neurons, nerve fibres, blood vessels and connective tissue. Two types of pinealocytes could be identified by light microscopy. They are large rounded with centrally placed nucleus, and small rounded pinealocytes. They have medium sized processes stainable with Alcian blue, periodic acid Schiff and Nissl methods. The pinealocytes are not stainable with bromophenol blue. However, they are moderately stainable with PAS, Sudan black and Baker's acid hematin. Neurons are seen either singly or in groups with axonal processes. Cystic cavities often lined by cells are a normal feature of adult squirrel pineal, and the lining cells are both pinealocytes and glial cells. Often neuronal endings are seen terminating on these lining cells. PAS positive globules were also seen inside the cysts. In some squirrel pineals, fibrous cysts with an inner core of cells are also seen. Occasionally groups of lymphocytes were also encountered in the pineal. In the fetal pineal, the cells are both larger and smaller ones and arranged in a cortex and medulla pattern and no cystic cavities are seen. The third ventricle enters the base of the pineal as pineal recess.     

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.     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。