Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice

Summary In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.     

Ultrastructural features of acidic complex carbohydrates in the intercellular matrix of the ovarian follicles in adult mice.

In the intercellular matrix of the granulosa layer of the mouse ovarian follicles, ultrastructural features of acidic complex carbohydrates have been studied by means of dialyzed iron (DI) staining in combination with procedures of digestion with Streptomyces and testicular hyaluronidases. In the intercellular matrix, DI reactive structures containing acidic complex carbohydrates consist of layers of a variable thickness coating the plasma membrane of the granulosa cells and reticular elements distributed in the spaces between the cells. The latter exists in two appearances; one is clumped masses of irregular shapes and different sizes, whereas the other being filamentous figures radiating from the masses. The effects of digestion with Streptomyces and testicular hyaluronidases upon the DI staining of the tissues indicate that the DI reactive structures in the intercellular matrix contain at least three types of acidic complex carbohydrates; hyaluronic acid, isomeric chondroitin sulfates and other acidic glycosaminoglycans. The histophysiological activities played by these particular complex carbohydrates have been briefly discussed.     

The histochemistry of complex carbohydrates in the scrotum of the boar

Summary In the scrotal skin of the boar, the histochemistry of complex carbohydrates has been studied by means of a series of selected methods of light microscopy. The epidermis of the scrotal skin was found to contain neutral and acidic complex carbohydrates with different saccharide residues. The secretory epithelial cells and secretory substances of the saccular apocrine sweat glands contained sulfated, other acidic and neutral complex carbohydrates, whereas the secretory epithelial cells and secretory substances of the tubular apocrine sweat glands involved largely neutral complex carbohydrates. The two types of complex carbohydrates from the both glands were shown to contain commonly substantial amounts of various saccharide residues but were devoid of notable amounts of sialic acid residues. In addition, complex carbohydrates in the smooth muscle cells were reacted for relatively small amounts of saccharide residues. From the present results, the histophysiological significanses of complex carbohydrates in the particular histologic structures of the scrotum have been discussed with special reference to the functions of the skin in the boar.A major part of this work has been presented at the 6th International Histochemistry and Cytochemistry Congress, Brighton, United Kingdom, in 1980     

Utilization of -glutamine in intercellular adhesion: Ascites tumor and embryonic cells

-Glutamine is required for the synthesis of complex carbohydrates required for the intercellular adhesion of mouse teratoma cells. It remained to be seen if these pathways were of general importance in the adhesion of other cell types. In this study, using an electronic particle counter assay to measure cell adhesion, Ehrlich ascites, Sarcoma 180 and Taper liver ascites tumor cells require exogenous -glutamine to aggregate. This effect is concentration dependent and the amino sugar, -glucosamine, replaces the glutamine requirement. Structural analogs of the active compounds are substantially less effective and metabolic inhibitors block the activity of the effective compounds. Two specific glutamine antagonists, DON (6-diazo-5-oxo- -norleucine) and azaserine (O-diazoacetyl-serine) decrease the action of -glutamine but not of -glucosamine. Trypsin dissociated six day old chick embryo neural retina cells do not require -glutamine to reaggregate, though the rate of aggregation is enhanced after preincubation with glutamine. Dissociation of small clumps of neural retina and inhibition of reaggregation of these cells are facilitated by preincubation with azaserine for 3–5 h. -Glutamine reduces the effect of azaserine on retina cells. These results are consistent with known metabolic pathways and suggest that -glutamine is involved in the synthesis of complex carbohydrates necessary for adhesion in a variety of cell types. The defective adhesion of the tumor cells examined may result from inability to produce glutamine synthetase, or effectively store cr transport -glutamine.     

Histochemistry of carbohydrates in the epithelium lining the bulbourethral gland of the rat

The histochemistry of carbohydrates has been studied in the epithelium lining the bulbourethral gland of the rat by means of light- and electron-microscopic methods. The results obtained show that the cytoplasms of the epithelial cells contain neutral and acidic carbohydrates. The neutral carbohydrates exhibited positive reactions with periodic acid-Schiff and periodic acid-thiosemicarbazide-silver proteinate, whereas the acidic carbohydrates reacted positively with alcian blue (AB; pH 1.0 and 2.5) and dialyzed iron. Most neutral carbohydrates were found to be glycoproteins localized within the secretory granules. The acidic carbohydrates consist of at least two types, AB (pH 1.0)-reactive sulfated and AB (pH 2.5)-reactive nonsulfated carbohydrates; most nonsulfated carbohydrates were determined to be sialic acid. The acidic carbohydrates were also localized within the secretory granules.     

The structure and cytochemistry of the neurosecretory cells in the intertidal gastropod Thais bufo (lamarck)

Three types of neurosecretory cells ('A', 'B' and 'C' cells) have been distinguished in the central ganglion of Thais bufo. A few homogenous groups are met with and the rest are all heterogenous groups. The histochemical observations reveal that the neurosecretory material is rich in carbohydrates, disulphides, protein bound amino groups, glycoprotein and lipids. Thus the neurosecretory material seems to be a lipoprotein--glycoprotein complex.     

Release of glycopeptides and mucopolysaccharides from ascites hepatoma cells by tryptic treatment.

Two types of ascites hepatoma cells, AH 66 and AH 130 FN, were treated with trypsin to observe the release of complex carbohydrates constituting the plasma membranes. From AH 66 cells, mucopolysaccharide (heparan sulfate) was preferentially released. From AH 130 FN cells, N-glycosidic glycopeptides were preferentially released whereas no mucopolysaccharide (chondroitin sulfate A) was released.     

Histochemistry of complex carbohydrates in the skin of the pig snout, with special reference to eccrine glands

The histochemistry of carbohydrates has been studied in the skin of the pig snout with selected methods of light microscopy including peroxidase-labelled lectin-diaminobenzidine (PO-LT-DAB) procedures. In the snout skin the dark secretory cells and the luminal secretion of the eccrine glands contained considerable amounts of neutral glycoproteins, but only a very small amount of acidic ones. It was possible to demonstrate glycogen in the secretory cells of these glands. The other skin structures of the snout also showed positive reactions for complex carbohydrates. Most remarkable were stronger reactions of intercellular substances among the spinosum cells, particularly following the PO-LT-DAB procedures, which demonstrated such saccharide residues as beta-D-galactose and N-acetyl-D-glucosamine.     

Cellular types in the gill arch of the teleost Cyprinus carpio (Cyprinidae, Pisces). A histochemical and ultrastructural study

On adult specimens of the common carp (Cyprinus carpio), we have carried out a histochemical and ultrastructural study of the epithelia which form the gill arch. Secondary lamellae have two cellular types, granular and mucous, which produce neutral carbohydrates and proteins rich in tryptophane, and another mucous cell type which contains glycosaminoglycans. In gill rakers, three cellular types show different histochemical behaviour: 1) granular cells elaborate neutral mucosubstances; 2) a second cell type contains glycosaminoglycans,, and 3) a third cell type secretes neutral and acid carbohydrates. We discuss the possible role of these cells according to their secretion. We describe pillar and chloride cells in secondary lamellae, and chloride and neuroendocrine-like cells in gill rakers.     

Polysaccharide processing and presentation by the MHCII pathway

The adaptive immune system functions through the combined action of antigen-presenting cells (APCs) and T cells. Specifically, class I major histocompatibility complex antigen presentation to CD8(+) T cells is limited to proteosome-generated peptides from intracellular pathogens while the class II (MHCII) endocytic pathway presents only proteolytic peptides from extracellular pathogens to CD4(+) T cells. Carbohydrates have been thought to stimulate immune responses independently of T cells; however, zwitterionic polysaccharides (ZPSs) from the capsules of some bacteria can activate CD4(+) T cells. Here we show that ZPSs are processed to low molecular weight carbohydrates by a nitric oxide-mediated mechanism and presented to T cells through the MHCII endocytic pathway. Furthermore, these carbohydrates bind to MHCII inside APCs for presentation to T cells. Our observations begin to elucidate the mechanisms by which some carbohydrates induce important immunologic responses through T cell activation, suggesting a fundamental shift in the MHCII presentation paradigm.     

Synthesis,conformation and T-helper cell stimulation of an O-linked glycopeptide epitope containing extended carbohydrate side-chains

To answer the question whether or not T cells to immunodominant protein fragments recognize glycosylated antigens, we synthesized a series of glycopeptides corresponding to peptide 31D, a major T-helper cell epitope of the rabies virus nucleoprotein. Thr4 of the epitope is known to allow mono- or disaccharide side-chain substitutions in either - or β-anomeric configuration without interfering with MHC-binding. To model naturally occurring glycoprotein fragments that carry extended sugar chains, we prepared Fmoc-Ser/Thr-OPfp building blocks containing - and β-linked linear tri- and heptasaccharides. Peptide 31D was synthesized with the complex carbohydrates attached to Thr4, and the T-helper cell activity of the glycopeptides was determined. Addition of -linked carbohydrates, that mimic most of the natural O-linked glycoproteins, resulted in a major drop in the T-cell stimulatory ability in a sugar length-dependent manner. In contrast, the cytosolic glycoprotein mimicking β-linked glycopeptides retained their T-cell stimulatory activity, with the trisaccharide-containing analogue being almost as potent as the unglycosylated peptide. When the peptides were preincubated with diluted human serum, all peptides lost their ability to stimulate the 9C5.D8-H hybridoma. These findings indicated that (i) in contrast to cytosolic glycosylation, incorporation of long O-linked carbohydrates into T-helper cell epitopes abrogates the antigenicity of these protein fragments, and (ii) glycosylation is not a viable alternative to improve the immunogenic properties of subunit peptide vaccines. Glycosylation with all four carbohydrate moieties similarly destroyed the inducible -helical structure of peptide 31D as detected by CD, indicating that the differences in the T-cell activity were not due to different peptide conformations.     

Secretory glycoconjugates in the epithelium of the goat prostate

Summary Secretory glycoconjugates in the epithelium of the goat prostate were investigated with various electron microscopical histochemical methods that involved periodic acid—thiocarbohydrazide—silver proteinate, colloidal gold-labelled lectin and related procedures. The three types of cells in the epithelium previously differentiated with the light microscope were substantiated: mucus-producing cells, protein-secreting cells and cells intermediate between both types. These three cell types contained varying amounts of neutral glycoconjugates; the histochemical nature of these carbohydrates was determined with particular emphasis upon the histophysiological functions of the accessory sex glands.     

Cell-type-related segregation of surface galactosyl-containing components at an early developmental stage in hemopoietic bone marrow cells in the rabbit

The avidin-biotin complex was used for the selective ultrastructural labeling of terminal cell surface galactosyl residues. Rabbit bone marrow cells were treated with the enzyme galactose oxidase in the presence of biotin hydrazide. Subsequent treatment with ferritin-avidin conjugates enabled the electron microscopic visualization of terminal membrane-based galactose and/or N-acetylgalactosamine on these cells. All stages of erythroid development were characterized by high levels of exposed cell surface galactose, whereas all leukoid cells in the same preparations were virtually unlabeled by the above method. Modulations in the distribution of these surface determinants during differentiation and maturation of rabbit erythroid cells were found to concur in inverse fashion with respect to that of terminal sialic acids. Neuraminidase treatment, before the above labeling procedure, resulted in the exposure of additional galactosyl residues on the surface of all bone marrow cell types. The results indicate that a galactose-bearing glycoconjugate(s) may comprise an erythroid-specific membrane constituent of rabbit bone marrow cells. The high density of galactose on the surface of even the earliest erythroid precursors may eventually enable the identification and isolation of a stem cell, which already contains the erythroid-specific galactoconjugate(s). The results suggest that variations in the spectrum of cell surface carbohydrates may serve as recognition signals in the complex set of intercellular interactions which occur during the development and maturation of the erythrocyte. The occurrence of similar but species- specific variations in the complement of surface heterosaccharides during erythroid development of humans and other mammals supports this contention.     

Histochemical analysis of glycoconjugates in the eccrine glands of the raccoon digital pads

The distribution and selectivity of complex carbohydrates in the eccrine glands of the digital pads in the North American raccoon (Procyon lotor) were studied using light and electron microscopic histochemical methods, particularly lectin histochemistry. In the eccrine glands, the dark cells exhibited neutral and acidic glycoconjugates with different saccharide residues (alpha-L-fucose, beta-D-galactose, beta-N-acetyl-D-glucosamine and N-acetyl-neuraminic acid); the clear cells contained numerous glycogen particles and showed a distinct reaction of alpha-L-fucose. The presence of complex carbohydrates with various terminal sugars was evident in the excretory duct cells. In addition, beta-D-galactose and N-acetyl-neuraminic acid residues were mainly observed in the luminal secretion. The glycoconjugates produced by the eccrine glands of the raccoon digital pads may protect the epidermis against physical damage or microbial contamination. In this way, the normal functioning of the sensory apparatus of the foot pads is ensured.     

Versatile derivatives of carbohydrate-binding modules for imaging of complex carbohydrates approaching the molecular level of resolution

The innate binding specificity of different carbohydrate-binding modules (CBMs) offers a versatile approach for mapping the chemistry and structure of surfaces that contain complex carbohydrates. We have employed the distinct recognition properties of a double His-tagged recombinant CBM tagged with semiconductor quantum dots for direct imaging of crystalline cellulose at the molecular level of resolution, using transmission and scanning transmission electron microscopy. In addition, three different types of CBMs from families 3, 6, and 20 that exhibit different carbohydrate specificities were each fused with either green fluorescent protein (GFP) or red fluorescent protein (RFP) and employed for double-labeling fluorescence microscopy studies of primary cell walls and various mixtures of complex carbohydrate target molecules. CBM probes can be used for characterizing both native complex carbohydrates and engineered biomaterials.     

Histochemical studies of acid proteoglycans and glycoproteins and activities of hydrolytic and oxidoreductive enzymes in the skin epidermis of the fish Blennius sanguinolentus pallas (Teleostei: Blenniidae)

In the epidermis of the fish Blennius sanguinolentus the histochemistry of complex carbohydrates and various oxidoreductases has been studied by means of a series of selected light microscopical techniques. The epidermis is endowed with three types of secretory cells namely, the mucous goblet cells, the superficial polygonal cells and the ionocytes, which provide protective functions in view of their involvement in the prevention of the skin epithelium from invading pathogens and in the osmoregulation processes respectively. The secretory substances of mucous goblet cells contain sulfated, carboxylated and neutral complex carbohydrates in addition to a glycoprotein with sialic acid terminal to galactose in its oligosaccharide chains. Activities of SDH, ICDH, MDH and G-6-PDH were studied, to elucidate some aspects of the correlated functions of the ionocytes which play a key role in the performance of the maintenance of the electrolyte pattern in the internal milieu of the skin. Differences in the intensity of various oxidoreductases are correlated with the extent of the activity of the epidermis for related to secretion of a mucous cover over the surface. Activity of the oxidoreductases is confined mainly to the basal and outer epidermal layers and this enzyme zonation is discussed with reference to the existence of the high turnover rate of the epithelial cells and both the proliferative and respiratory capabilities of the skin epithelium.     

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.     

The histochemistry of complex carbohydrates in the testes of patients of idiopathic male infertility

Summary In testis tissues from patients with idiopathic male infertility, complex carbohydrates have been studied by means of light microscopic histochemical methods, comparisons being made with the normal testes. In the infertile testes, histochemical reactions for acidic and neutral complex carbohydrates were in general weaker in intensity than in the normal testes. In the seminiferous tubules and interstitium of the infertile testes, a histochemical finding of primary importance was obtained with the demonstration of galactose deficiency in the complex carbohydrates involved. These glycoproteins contained only small numbers of sulfhydryl and disulfide groups. The possible histophysiological significance of these complex carbohydrates is discussed, with special reference to their histochemical properties determined in the present study.     

The histochemistry of complex carbohydrates in the testes of patients of idiopathic male infertility.

In testis tissues from patients with idiopathic male infertility, complex carbohydrates have been studied by means of light microscopic histochemical methods, comparisons being made with the normal testes. In the infertile testes, histochemical reactions for acidic and neutral complex carbohydrates were in general weaker in intensity than in the normal testes. In the seminiferous tubules and interstitium of the infertile testes, a histochemical finding of primary importance was obtained with the demonstration of galactose deficiency in the complex carbohydrates involved. These glycoproteins contained only small numbers of sulfhydryl and disulfide groups. The possible histophysiological significance of these complex carbohydrates is discussed, with special reference to their histochemical properties determined in the present study.     

Relationship of adhesiveness of cells in culture with specific enzyme activity.

l-Glutamine is required by mouse teratoma cells and other mouse ascites tumor cells in the synthesis of complex carbohydrates involved in intercellular adhesion. Since l-glutamine is synthesized by the enzyme glutamine synthetase (GS) (EC 6.3.1.2), these studies were undertaken to determine if a relationship exists between cellular adhesiveness and GS specific activity. Two types of experiment were performed to examine this relationship. Actinomycin D enhanced both teratoma cell GS specific activity and cellular adhesiveness over controls in batch cultures at confluency. Also, the relationship between cell adhesiveness and GS specific activity during the cell cycle was studied using cell populations synchronized with thymidine plus Colcemid. In these synchronized cultures, cellular adhesiveness displayed an oscillatory pattern with peaks of GS specific activity occurring just prior to peaks of adhesiveness. The levels of GS specific activity and intercellular adhesiveness were enhanced by the addition of hydrocortisone, a steroid known to induce GS specific activity in mouse teratoma cells. These results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implicates l-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.