Optical polarization reveals different ultrastructural molecular arrangement of polysaccharides in the yeast cell walls.

The topo-optical aldehyde bisulfite-toluidine blue (ABT) reaction of vicinal OH and amino-OH groups offers new ways to study the ultrastructure of polysaccharides in different biological substrates. Through oriented dye binding on the reacting groups, the ABT reaction induces strong birefringence on the linearly ordered polysaccharides, which is negative with respect to their chain length. Using this method, two types of molecular order of the polysaccharides could be distinguished in the cell walls and capsules of yeasts. (1) The optically negative spherulitic character of the yeasts after the ABT reaction indicated that the toluidine blue molecules were bound tangentially (in a surface-parallel pattern) while the polysaccharide chains of the cell walls and capsules were oriented mainly radially. This structural pattern may be explained as resulting from a helicoid conformation of the polysaccharide component. (2) Acid or alkali hydrolysis removed the radially oriented polysaccharide component of the cell wall. The remaining, resistant polysaccharides showed up in the form of optically positive spherulites indicating radially oriented dye molecules on a circularly ordered, micellar polysaccharide texture.     

Demonstration of microorganisms in tissues by the ABT and KOH-ABT topo-optical reactions.

The aldehyde-bisulphite-toluidine blue (ABT) reaction, as a selective topo-optical test of vicinal OH and amino-OH groups is suited for the selective demonstration in tissues of microorganisms of polysaccharide containing cells. Alkaline pretreatment of the polysaccharide cell walls, releases, by splitting the O-acyl radicals, further vicinal OH groups for the ABT reaction, thus actually increases the sensitivity of the method. The topo-optical reactions are characterized by a strong birefringence induced by oriented dye-binding, due to the linear arrangement of polysaccharides composing the cell wall. Differences in the character of birefringence have made it possible to work out a new method for the analysis of the cell wall ultrastructure as well as to demonstrate microorganisms in tissues. The practical value of the reactions is illustrated by examples.     

Sulfation as a collagen-specific reaction The ultrastructure of sulfate collagen,basement membranes and reticulin fibers as shown by topo-optical staining reactions

Summary Sulfation induces hyperbasophilia in connective tissue structures (fibrillary collagen, basement membranes and reticulin fibers), which appear metachromatic with toluidine blue at pH 1.0 and strongly birefringent with inversion of their positive birefringence into negative birefringence indicating transversally oriented and closely packed dye molecules on the micellar surface of collagen. Quantitative studies of the sulfation induced topooptical staining reaction following blocking of the vicinal glycol groups by periodate and the enzymatic removal of AMP support the view that carbohydrate glycol groups play only a minor part and the OH side-groups of the collagen peptide chains play the major part in the sulfation reaction of fibrillary collagen and basement membranes.After blocking of the vicinal glycol groups of carbohydrate components by periodate, sulfation induced toluidine blue hyperbasophilia with strong negative birefringence associated with selective proteolytic sensitivity are collagen-specific characeteristics due to sulfate esterification on the OH groups of the peptide chains of collagen, which provide new approach to the study of the ultrastructure of connective tissue elements in physiology and pathology.     

Ultrastructural visualization of carbohydrates in oxytalan fibers in monkey periodontal ligaments

Fullmer's oxytalan fibers appear to be special connective tissue fibers belonging to elastic system fibers. We have ultrastructurally examined carbohydrates in oxytalan fibers in monkey periodontal ligaments after glutaraldehyde fixation and ethylenediaminetetraacetic acid (EDTA) decalcification using: Thiéry's periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) method for thin-section staining of vicinal glycol-containing complex carbohydrates, and the concanavalin A-ferritin (Con A-ferritin) and Con A-horseradish peroxidase (Con-A-HRP) en bloc staining methods specific for alpha-D-mannosyl and alpha-D-glucosyl groups. PA-TCH-SP stained collagen fibrils weakly to moderately and stained oxytalan fibers moderately. Con A-ferritin and Con A-HRP stained collagen fibrils weakly or moderately and stained oxytalan fibers intensely within the superficial region of specimen blocks. The penetration of staining reagents was improved by prior saponin treatment and/or chondroitinase ABC digestion. Thus, these studies demonstrate that PA-TCH-SP and Con A staining of carbohydrates is very useful in identifying oxytalan fibers at the ultrastructural level and that more carbohydrate components are present in oxytalan fibers than in collagen fibrils.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358 +/- 0.134 X 10(4) at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.     

Acid polysaccharide content of frog rod outer segments determined by metachromatic toluidine blue staining

Summary Sulfonation of periodate-oxidized vicinal hydroxyl groups on a polysaccharide backbone allows binding of toluidine blue (aldehyde bisulfite-toluidine blue or ABT staining) with a concurrent metachromatic shift of the dye's absorption peak from 630 nm (monomer) to 580 nm (isolated dimer interaction at vicinal sulfonate groups) or 540 nm (dye polymer interaction). A molar absorptivity of 2.358±0.134×10⁴ at 540 nm for polymeric toluidine blue O chloride (TB) aggregates was determined by spectrophotometry of TB bound to hyaluronic acid (HA) and sulfonated glycogen (SG) in water. Microspectrophotometry of ABT stained frog rod outer segments (FROS) showed spectra similar to TB in aqueous HA and SG solutions with absorbances corresponding to 0.063 M dye bound to sugar. Given two dye molecules bound per sugar residue and a rhodopsin concentration of 3.25 mM in FROS, the above indicates 10 stainable sugars per rhodopsin are contained in these cells. Half of these sugars are sensitive to hyaluronidase digestion implying 5 glycosaminoglycan (GAG) repeating units and 5 stainable oligosaccharide sugar residues per rhodopsin in FROS. The GAGs in FROS appear to be primarily HA. Birefringence measurements at 475 nm indicate that this HA and the oligosaccharide of rhodopsin are anisotropically oriented in these cells.Supported by NIH grants EY00012, EY07035 and EY01583     

Polarization optical analysis of blood cell membranes

The present study deals with investigations of membrane structure using polarization topo-optical reactions. Polarization microscopy is a special field of biological submicroscopic morphology. It represents a powerful tool well able to reveal the features of organization of biological structures, and the regularity of macromolecules building cells and tissues - properties that cannot directly be studied by other approaches to complex biological systems. Only in "pure" systems can X-ray diffraction, or the analysis of circular dichroism and the dispersion of optical rotability provide data equivalent to those obtained by polarization microscopy in complex systems. One of the main drawbacks of molecular biology is that most information is relevant to isolated, purified particles or macromolecules. Thus, no conclusions can be drawn concerning the original arrangement of molecules. The gap between biochemical-biophysical and morphological approaches to molecular arrangement in complex structures is bridged by the polarization optical technique. As was pointed out in the introduction, polarization microscopy became a routine biological research method following the pioneering work of Romhányi. His enlightening topo-optical reactions (Romhányi 1960, 1963, 1966) were based on the oriented dye binding of the original charge carriers of regularly arranged tissue constituents. The second group of Romhányi's topo-optical reactions comprised procedures such as sulfation (Romhányi et al. 1973, 1974), the aldehyde-bisulfite-toluidine blue (ABT) reaction (Romhányi et al. 1974, 1975), the permanganate-bisulfite-toluidine blue (PBT) reaction (Fischer 1979, 1979a), and the sialic acid-specific reaction (Makovitzky 1980) all of which operate with induced dye-binding groups; i.e. dye-binding moieties on biological macromolecules are produced by specific chemical reactions.     

Histochemical differentiation of complex carbohydrates with variants of the concanavalin A-horseradish peroxidase method.

Various treatments carried out prior to the concanavalin A-horseradish perioxidase (HRP) method have been found to affect the staining and have permitted differentiation of three main classes of complex carbohydrates in the rat alimentary tract. Class I mucosubstances lose and class II and III paradoxically gain concanavalin A-horseradish peroxidase reactivity after periodate oxidation. Class II mucosubstances lose whereas class III retain or increase their reactivity with a reduction step interposed between oxidation and concanavalin A-horseradish peroxidase staining. Mucous neck cells, pyloric glands, Brunner's glands and mast cells exhibit strong class III staining, whereas other sites such as intestinal goblet and salivary gland acini differ widely in their type of staining. Liver glycogen stains like mucosubstances in an unstable subgroup of class III. The paradoxical increase in concanavalin A binding during oxidation correlates with the appearance of Schiff reactivity implicating oxidation of vicinal hydroxyls as the basis for the effect. The periodate-induced staining is therefore, thought to result from an oxidative disruption of linkages between vicinal hydroxyls of neighboring sugars and hydroxyls of mannose required for concanavalin A binding. Staining with the described concanavalin A-horseradish peroxidase variants appears to afford information concerning cytochemical distribution of mannose-rich glycoproteins as well as differences among these substances in the relation of mannose to neighboring sugars.     

Mapping Molecular Association Networks of Nervous System Diseases via Large-Scale Analysis of Published Research

Network medicine has been applied successfully to elicit the structure of large-scale molecular interaction networks. Its main proponents have claimed that this approach to integrative medical investigation should make it possible to identify functional modules of interacting molecular biological units as well as interactions themselves. This paper takes a significant step in this direction. Based on a large-scale analysis of the nervous system molecular medicine literature, this study analyzes and visualizes the complex structure of associations between diseases on the one hand and all types of molecular substances on the other. From this analysis it then identifies functional co-association groups consisting of several types of molecular substances, each consisting of substances that exhibit a pattern of frequent co-association with similar diseases. These groups in turn exhibit interlinking in a complex pattern, suggesting that such complex interactions between functional molecular modules may play a role in disease etiology. We find that the patterns exhibited by the networks of disease – molecular substance associations studied here correspond well to a number of recently published research results, and that the groups of molecular substances identified by statistical analysis of these networks do appear to be interesting groups of molecular substances that are interconnected in identifiable and interpretable ways. Our results not only demonstrate that networks are a convenient framework to analyze and visualize large-scale, complex relationships among molecular networks and diseases, but may also provide a conceptual basis for bridging gaps in experimental and theoretical knowledge.     

Ultrastructural cytochemistry of complex carbohydrates in developing feline neutrophils

The feline species provides animal models for at least six congenital lysosomal disorders. Since knowledge of normal feline neutrophils is a prerequisite for studies of their abnormalities, the present report describes the morphology and cytochemistry of normal feline neutrophils and compares the subcellular distribution of sulfate- and vicinal-glycol-containing complex carbohydrates to that of peroxidase and acid phosphatase. Immature feline primary granules, formed in promyelocytes, were stained for peroxidase, acid phosphatase, sulfate, and vicinal glycols. During maturation, primary granules retained strong staining for peroxidase, but staining for vicinal glycols decreased, and acid phosphatase and sulfate reactivity was lost. Secondary granules formed in myelocytes lacked peroxidase, acid phosphatase, and sulfate staining, but stained intensely for vicinal-glycol-containing complex carbohydrates. No analogues of tertiary granules previously described in rabbits and humans were demonstrated in feline neutrophils. However, a new sequential staining technique for peroxidase and vicinal glycols has suggested the formation in myelocytes and late neutrophils of a third granule type that contained peroxidase, acid phosphatase, and vicinal glycols but lacked sulfate staining. Thus, the staining characteristics of primary and secondary granules in cats closely resembled those in humans and rabbits. The third (late-forming) type of granule has not previously been described in other species.     

Selective Recovery of Carbohydrates from Aqueous Solution Facilitated by a Carrier

Carbohydrate recovery is an active area of supramolecular chemistry, motivated by the biological importance of saccharides as well as the unusual challenge presented by these complex substances. The recovery of carbohydrates from aqueous media is a difficult separation problem due to the large, irregular and multivalent structure and the low solubility of carbohydrates in organic solvents. A method for the selective recovery of mono‐ and disaccharides from aqueous media has been developed. The use of different organic solvents like butanol, methyl tertiary‐butyl ether (MTBE), n‐hexane or toluene for liquid‐liquid extraction of carbohydrates was investigated. This extraction process is facilitated by a carrier, i.e., primary amines such as cyclooctylamine. The influence of different parameters (temperature, amine concentration, extraction time) on the efficiency of the extraction was studied. Recovery rates up to 40 % are possible in a one‐stage process. Selectivities range from 1.3 up to 875.4.     

Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review

A review concerning the definition, extraction, characterization, production and functions of extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment reactors is given in this paper. EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms, produced from cell lysis and adsorbed organic matter from wastewater. They are a major component in microbial aggregates for keeping them together in a three-dimensional matrix. Their characteristics (e.g., adsorption abilities, biodegradability and hydrophilicity/hydrophobicity) and the contents of the main components (e.g., carbohydrates, proteins, humic substances and nucleic acids) in EPS are found to crucially affect the properties of microbial aggregates, such as mass transfer, surface characteristics, adsorption ability, stability, the formation of microbial aggregates etc. However, as EPS are very complex, the knowledge regarding EPS is far from complete and much work is still required to fully understand their precise roles in the biological treatment process.     

Phenylarsine oxide and the mechanism of insulin-stimulated sugar transport

The actions of phenylarsine oxide (PAO) on hormone receptors and transport processes are reviewed with particular reference to the mechanism of insulin-stimulated sugar transport. It is suggested that as well as reaction with vicinal -SH groups, vicinal -SH/-OH and -SH/-CO2H groups should also be considered as potential reaction sites for PAO. The relatively high levels of these vicinal combinations of groups in many hormone receptors makes them particularly susceptible to reaction with PAO. In the case of insulin-stimulated sugar transport PAO does not inhibit insulin binding to its receptor at low concentrations but may react directly with the glucose transporters in some cells. A hypothesis is proposed suggesting that PAO may react specifically with one transporter isoform (GLUT-4) which is found almost exclusively in rat adipocytes, skeletal muscle and heart tissue (i.e. insulin responsive tissue) whereas in insulin unresponsive cells such as erythrocytes the GLUT-1 isoform is the predominant transporter which is not inhibited by PAO.     

Glycation of lysine-containing dipeptides.

Protein glycation through Maillard reaction (MR) is a fundamental reaction both in foods and in the human body. The first step of the reaction is the formation of Amadori product (AP) that is converted into intermediate and advanced MR products during reaction development. Although the MR is not an enzymatic reaction, a certain degree of specificity in the glycation site has been observed. In the present study, we have monitored the glycation of different lysine-containing dipeptides to evaluate the influence on the NH(2) reactivity of the neighboring amino acid.Lysine dipeptides were reacted with glucose, galactose, lactose and maltose. The formation and identification of glycated compounds were monitored by mass spectrometry (MALDI-TOF and ESI-MS/MS) and by HPLC of their Fmoc derivatives. MS/MS analysis showed that the glucose APs formed on dipeptides have a characteristic fragmentation pattern: the fragment at [M - 84](+) due to the formation of pyrylium and furylium ion is mainly present in the monoglucosylated form, while the [M - 162](+) and the [M - 324](+) are more evident in the fragmentation pattern of the diglucosylated forms.The nature of the vicinal amino acids strongly affects lysine reactivity towards the different carbohydrates: the presence of hydrophobic residues such as Ile, Leu, Phe strongly increases lysine reactivity. Contrasting results were obtained with basic residues. The Lys-Arg dipeptide was among the most reactive while the Lys-Lys was not.     

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     

High-performance liquid chromatography of monosaccharides and oligosaccharides in a complex biological matrix.

Analysis of oligosaccharides in complex biological matrices is hampered by the fact that oligosaccharides, closely related in structure, are difficult to separate from each other and that conventional detection procedures (refraction index and uv detection) are not specific enough for carbohydrates. Prepurification of samples by procedures like desalting or gel filtration is often used but can lead to the loss of specific oligosaccharides. We have used pellicular anion chromatography in combination with a postcolumn reaction for reducing carbohydrates based on 4-aminobenzoylhydrazide. This procedure not only detected normal mono- and oligosaccharides but N-acetylhexosamines and reducing N-acetylhexosamine containing oligosaccharides as well. A sensitivity of about 20-25 pmol for non-GlcNAc containing mono- or oligosaccharides and between 30-50 pmol for GlcNAc or oligosaccharides with GlcNAc at the reducing side was reached. The postcolumn detection was compared with pulsed amperometric detection and appeared to be more specific for mono- and oligosaccharides. Except for deproteination to protect the column, no further sample preparation was needed with this system for our application (urines). In this way pellicular anion chromatography in combination with this postcolumn reaction reaction to be a sensitive and specific HPLC procedure for analysis of monosaccharides and oligosaccharides in complex biological matrices.     

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.     

A new lectin-gold complex for ultrastructural localization of galacturonic acids

We report the development of a cytochemical affinity technique for detection of galacturonic acids at the ultrastructural level. The highly purified gonad lectin from Aplysia depilans (AGL) was tagged with colloidal gold particles and used for labeling carbohydrates in resin-embedded sections of various plant and fungal tissues. Patterns of AGL binding sites were compared to those obtained with a D-galactose-specific lectin, Ricinus communis agglutinin I. Differences in labeling patterns were noted, indicating that the lectins exhibited differential carbohydrate binding. In addition, the considerable loss of labeling over isolated wheat coleoptile walls treated for removal of pectin, after incubation with the AGL-gold complex, strongly suggested an affinity of AGL for pectic substances. A series of cytochemical controls, including sugar inhibition tests, has proven the specificity of the technique and the high affinity of AGL towards galacturonic acids. The potential value of this new lectin for ultrastructural studies on cell wall pectic substances in plant biology and pathology is demonstrated.     

Relative reactivity of hydroxyl groups in inositol derivatives: role of metal ion chelation

O-Alkylation of myo-inositol derivatives containing more than one hydroxyl group via their alkali metal alkoxides (sodium or lithium) preferentially occurs at a hydroxyl group having a vicinal cis-oxygen atom. In general the observed selectivity is relatively higher for lithium alkoxides than for the corresponding sodium alkoxide. The observed regioselectivity is also dependent on other factors such as the solvent and reaction temperature. A perusal of the results presented in this article as well as those available in the literature suggests that chelation of metal ions by inositol derivatives plays a significant role in the observed regioselectivity. Steric factors associated with the axial or equatorial disposition of the reacting hydroxyl groups do not contribute much to the outcome of these O-alkylation reactions. These results could serve as guidelines in planning synthetic strategies involving other carbohydrates and their derivatives.