Vic-glycol Groups Research Articles

Cytochemical characterization of the third-stage larva of Wuchereria bancrofti (Nematoda: Filarioidea)

In the present paper, we report the results we obtained using several cytochemical techniques to analyze the infective larva of Wuchereria bancrofti. An imidazole osmium tetroxide solution was used to visualize unsaturated fatty acids. A highly contrasted material forming a continuous structure was observed on the larval surface and over the epicuticle. A strong reaction was observed on the esophagus and also on the inner secreted material. Carbohydrates containing vic-glycol groups were not observed on the cuticle of the third-stage larva of W. bancrofti submitted to the Thiéry technique. Using a panel of eight gold-labeled lectins, we found that the cuticle exhibited slight labeling with all lectins used, indicating residues of N-acetyl-D: -glucosamine, N-acetyl-galactosamine, D: -galactose, D: -manose, and L: -fucose. Surface anionic sites were visualized by using cationized ferritin particles. Treatment with trypsin partially inhibited the reaction, whereas the treatment with chondroitinase ABC, a specific enzyme for glycosaminoglycans, completely abolished the labeling with cationic particles.

Role of Morphological Methods in the Analysis of Chemically Induced Colon Cancer in Rats

The role of selected histopathological methods was evaluated for the characterization of tumourous tissues resulting from chemically induced carcinogenesis in rats. Colon cancer was induced by treating rats with a direct carcinogen, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), alone or with subsequent treatments with secondary bile acids. Morphological studies showed that the tumorigenic effect of MNNG resulted in changes in the shape of the crypts, disappearance of mucous (goblet) cells, and marked increases in the mitotic index, the size of nuclei and the number of aberrant nuclei. A significant number of lymphocytes infiltrated between the crypts and into their lumens. A sharp decrease in concentrations of neutral mucopolysaccharides and sulfomucins and an increase in the amount of vic-glycol groups and nonsulfated mucosubstances were demonstrated in tumourous epithelial cells. A moderate positive reaction to tissue polypeptide antibody was observed in these cells. The tumor-promoting effects of secondary bile acids were detected morphologically by an increase in the amount of connective tissue that infiltrated newly formed tumors. The increased number of micronuclei in otherwise histologically unaltered colon tissues adjacent to a tumor suggests that these regions represent a precancerous stage in the development of tumors. The important role of morphological methods in the evaluation of the effects of carcinogen and tumor promoters and in the detection of various phases in experimental carcinogenesis was discussed.

Effect of tunicamycin on glycogen accumulation in the stratum intermedium and odontoblasts of rat incisor.

Repeated injection of rats with tunicamycin over two days induced a 1- to 5-fold increase in glycogen. This accumulation occurred in the stratum intermedium of the enamel organ and in young secretory odontoblasts. In rats injected over 3 days, the number of glycogen particles was at least 10 times larger than in control rats, and large glycogen accumulations were observed in the cytosol of these two groups of cells. These results were obtained by staining with periodic acid-thiocarbohydrazide and silver proteinate, a specific method for the detection of glycoconjugates containing vic-glycol groups. The existence of a relationship between these local cytosolic accumulations of glycogen and the developmental stage of certain groups of cells was shown by the changes that occurred in glycogen distribution. The present results suggest that the stratum intermedium supplies energy for precursor transport.

The Ultrastructure of the Organic Phase Associated with the Inorganic Substance in Calcified Tissues

An organic phase is closely associated with the mineral substance is all calcified matrices, where it can be demonstrated as crystal-bound proteins by biochemical methods and as crystal ghosts by electron microscopy. Interest in crystal ghosts derives chiefly from the observation that they have the same shape, arrangement, and orientation as inorganic crystallites, which suggests they may have a role in their formation. Histochemically, crystal ghosts of epiphyseal cartilage react with colloidal iron (pH 2.0), acidic phosphotungstic acid, ruthenium red, and a number of cations including calcium, barium, magnesium, lanthanum, strontium, and terbium chloride. Their reactivity is removed by methylation and only incompletely restored by saponification. Moreover, the crystal ghosts located at the periphery of the calcified areas contain vic-glycol groups, as shown by their reactivity with periodic acid-silver nitrate and periodic acid-thiosemicarbazide-osmium. All these reactions show that the crystal ghosts of epiphyseal cartilage contain acidic, probably sulfate groups and, at least initially, vic-glycol groups. Their reactivity decreases as the calcification process is completed. Although the available data are not sufficient to allow a full understanding of the nature and function of these structures, they seem to play an important role in calcification. The hypothesis is presented that crystal ghosts are preformed in calcifying matrices and are activated by the unmasking of the reactive groups in their polymeric molecule; the unmasked groups then link up with inorganic ions in such a way to form organic-inorganic structures the inorganic ions of which are arranged in an apatitelike configuration and the filamentlike shape of which is the same as that of the polymeric molecule.

The glucuronoxylans and the helicoidal shift in cellulose microfibrils in linden wood: Cytochemistryin muro and on isolated molecules

In fibres of wood, the classical S1 and S2 layers are connectedvia a transition zone where a helicoidal texture occurs. In order to understand the actual mechanism of cellulose microfibril rotation in this zone, the study of relationship between cellulose and matrix was undertaken cytochemically at the ultrastructural level. Glucuronoxylans,i.e., the main hemicellulose component of hardwood, were studied in cell walls of linden tree. Xylanase-gold complexes were used as a new cytochemical tool to directly and specifically label glucuronoxylans within the wall of fibres. Subtractive localization (KOH or DMSO extraction and PATAg test or shadowing) associated with chemical analysis was carried out as control. The study of isolated glucuronoxylan molecules was undertaken in parallel. Both from direct (xylanase-gold labeling) and indirect techniques (extractions), glucuronoxylans appear preferentially concentrated in the transition zone which overlaps the layers S1 and S2. A comparison between KOH and DMSO extraction indicates a difference in accessibility of glucuronoxylans distributed across the whole wall and those located in the transition zone. Isolated molecules have a rodlike aspect and show a tendancy to spatially organize in parallel alignment. Cytochemical labeling of the isolated molecules concerns covalent linkages, vic-glycol groups and acid side groups along the main chain. The preferential localization indicates that in the helicoidal zone glucuronoxylans constitute a thick matrix embedding the cellulose microfibrils in the course of rotation. This data leads to a discussion of how these localized matrix molecules could intervene in the assembly and the twisted morphogenesis of the fibre cell wall.

The presence of ethylenic bonds and vic-glycol groups in neuromelanin and lipofuscin in the human brain.

Through use of oxidation and blockading reactions, chemical group precursors of aldehyde demonstrable with Schiff reaction staining were identified in the soluble lipid-free lipofuscin component of neuromelanin of human substantia nigra and in lipofuscin of human inferior olive. Aldehyde generation was implied by moderate staining after bleaching neuromelanin and oxidizing lipofuscin with potassium permanganate followed by oxalic acid. Confirmation of aldehyde generation was achieved when diminished staining followed a sulfite addition blockade obtained by replacing oxalic acid with metabisulfite or bisulfite as well as by condensation blockades obtained with phenylhydrazine or aniline without replacing oxalic acid. Vic-glycol precursors of aldehyde were demonstrated in both pigments when acetylation or bromination preceded permanganate-oxalic acid and staining was unequivocally diminished only after acetylation. Vic-glycols were also demonstrated in lipofuscin by diminished staining when acetylation preceded periodic acid oxidation. Ethylenic precursors of aldehyde were suggested in performic acid-bleached neuromelanin when the minimal staining that followed this peracid's generation of Schiff reaction-negative dihydroxy groups became greatly intensified following an additional oxidation with periodic acid. This additional oxidation converted the dihydroxys to Schiff reaction-positive aldehyde. Ethylenes in lipofuscin were indicated when bromination before performic acid reduced subsequent staining.

A Cytochemical Investigation of the Lateral Walls of Dianthus Vessels. Differentiation and Pit-Membrane Formation

The deposition of cell wall polysaccharides and subsequent lignin incrustation were followed during vessel differentiation in carnation stems. In the secondary walls, lignin deposition was closely accompanied by an apparent loss of free vic-glycol groups from polysaccharides. This apparent loss could be related to the progressive binding of lignin molecules to cell wall carbohydrates. The intensity of lignification in mature intervessel primary walls exhibited a positive gradient from the earliest- to the latestformed metaxylem elements. Intervessel pitmembrane hydrolysis appeared as a sequential and ordered process. A partial hydrolysis of the vessel primary wall was sometimes observed in vessel-to-parenchyma pits.

Organization of the sieve-element walls of leaf minor veins

The organization of the sieve-element wall in minor veins of Beta vulgaris and 13 other species has been investigated at the electron microscope level using histochemical techniques. In all species examined, chemically distinct regions of the wall could be identified using the silver methanamine technique for staining carbohydrates with VIC-glycol groups and by phosphotungstic acid (PTA)/HCl poststaining. There appears to be a thin, dense layer of nonmicrofibrillar material adjacent to the sieve-element plasma membrane of most species examined. Cell-wall enzymatic digestions suggest this layer may have a high pectin content. This layer does not appear to be related to the often-reported nacreous layer of the inner sieve-element wall. Immediately surrounding this layer is a thick microfibrillar region with the microfibrils apparently transversely aligned with respect to the long axis of the cell. These two layers are interpreted as constituting the primary wall of the sieve element. The adjacent companion cell wall, which is clearly delineated by use of PTA/HCl staining, is thinner and of a different chemical composition compared with the sieve-element wall, with hemicellulose possibly predominating. Variations in cell wall structure among species are illustrated, including the wall structure of two species containing phloem transfer cells. The possible functions of the specialized wall structure are discussed.

The histochemistry and ultrastructure of the glandular vas deferens of Tubifex tubifex (Annelida: Oligochaeta)

The ultrastructure and histochemistry of the atrium, prostate gland and penis of Tubifex tubifex are described. The prostate gland, which forms a discrete structure alongside the atrium, consists of club‐shaped gland cells with narrow cell processes that converge onto the junction with the atrium, Prostate perikarya contain abundant rough endoplasmic reticulum (rER) and active Golgi cisternae and function in the synthesis of secretion granules. Prostate cell processes, characterized by a peripheral band of microtubules linked to an electron lucent cisterna derived from the perikaryon rER, penetrate the atrium and ramify between the atrial epithelium and musculature. The atrial epithelium consists of interstitial ciliated cells and anucleate glandular clements packed with secretion granules. Prostate cell processes ultimately open into the bases of the atrial glandular elements which therefore constitute the expanded apices of prostate cells. The morphological differentiation of prostate atrial gland cells into distinct zones concerned with the production, translocation and storage of secretion granules is discussed in relation to sperm transmission. The presence of a conventional peritoneum surrounding the prostate gland indicates that the gland cells originate from the atrial epithelium.The prostate‐atrial gland cells are of two types characterized by the ultrastructure and histochemistry of their secretion granules. Type I granules are uniformly electron dense and are composed of neutral mucopolysaccharide with vic glycol groups and basic protein containing tyrosine, tryptophan and cystine. Type I1 granules are heterogeneous and consist of electron dense subunits of basic protein within a microfibrillate matrix composed of carboxylated acid mucopolysaccharide. The possible function of prostatic secretion is discussed.The penis is composed of a simple tubular extension of the epithelial, muscular and peritoneal cells of the male duct‐body wall junction and is enclosed within a double foldin the epidermis. The penis lumen is plugged by secretion derived from the prostate‐atrial gland cells.

Histochemical studies of mucosubstances and keratins of thymic corpuscles.

Keratin and prekeratin have been visualized histochemically by testing the reactivity of disulfide groups in the thymic corpuscles of the sheep. Rhesus monkey, rabbit and dog, and the reactivity of sulfhydryl groups in the sheep, Rhesus monkey, rabbit and rat. Ten to 50% of the thymic corpuscles showed keratinization by this method. Acidic mucosubstances were visualized histochemically in the Rhesus monkey, sheep and rat but not in the rabbit. Staining for vic-glycol groups was prominent in all species tested. Neuraminidase treatment with subsequent Alcian blue-periodic acid-Schiff (PAS) staining indicated the presence of sialomucins in the thymic corpuscles of the monkey and rat, but sialomucins were not demonstrated in the rabbit.

Cytochemical Localization of Polysaccharides in Diplodia Maydis With Thiosemicarbazide : Evaluation of Three Fixatives For Pa:Tsc:Os Reaction

In an attempt to identify polysaccharide components in thin sections of D. maydis, procedures were employed such that a PAS localization could be carried out. Three different fixatives were evaluated ie. glutaraldehyde, formaldehyde and paraformaldehyde. These were used in conjunction with periodic acid (PA), thiosemicarbazide(TSC), and osmium tetroxide(Os) to localize polysaccharides in V. maydis using a pre-embedded reaction procedure. Polysaccharide localization is based on the oxidation of vic-glycol groups by PA, and the binding of TSC as a selective reaction center for the formation of osmium black. The reaction product is sufficiently electron opaque, insoluble in lipids, not altered when tissue is embedded, and has a fine amorphous character.

HISTOCHEMICAL STUDY OF A DERMATOPHYTE UNDER AN ELECTRON MICROSCOPE

Whichever sections of Trichophyton mentagrophytes were treated by chromic acid-silver methenamine, periodic acid-silver methenamine, periodic acid-thiocarbohydrazide-silver proteinate, or alkaline bismuth histochemical staining techniques, the cell walls were densely impregnated and clearly defined.In the case of sections stained with alkaline bismuth, the cell walls revealed histochemical ultrastructures, there being fine granular structures all over, microfibrous structures in the inner half of the cell wall, and coarse granular structures arranged close together on the outer surface of the plasma membrane.It was discovered from the results of controlled experiments involving acetylation, saponification, periodic acid oxidation and chitinase digestion that the chemical substances of the afore-mentioned structures are in the main composed of polysaccharides containing vic-glycol groups, and supported by chitin. Furthermore, satisfactory electron micrographs of the general structures of fungal cells were obtained from sections which had been treated with alkaline bismuth, combined with uranyl acetate, followed by the lead citrate poststaining technique.

A NEW ELECTRON MICROSCOPIC HISTO-CYTOCHEMICAL STAINING METHOD -DEMONSTRATION OF GLYCOGEN PARTICLES-

For electron microscopic demonstration of glycogen particles in tissue sections, a new histochemical staining method is introduced as a substitute for stains such as periodic acid thiosemicarbazide silver proteinate. It is based on the fact that glycogen particles can be demonstrated with alkaline bismuth (A·Bi) solution alone and without periodic acid pretreatment.A. The staining mechanism involves a direct reaction where vic-glycol groups in glycogen compounds are oxidized into carboxyl groups and bismuth is reduced. The latter combines with the carboxyl groups to effect increased electron opacity of glycogen particles under electron microscopic observations.B. The glycogen particles can be observed even if periodic acid treatment is added on condition that the treatment be stopped within 10-15min. But glycogen particles disappear when the treatment lasts more than 30min. The treatment is not necessary even in theory because the A·Bi stainable vic-glycol groups are gradually decreased in proportion to the duration of the treatment.C. The technique is adapted to the observation under high magnification of the mode of α-D-glucopyranose arrangement in glycogen compounds because the bismuth deposit has a high electron opacity due to the high atomic number of bismuth and is positioned only in the vic-glycol group of the glycogen compound.D. The staining technique is remarkably simple as staining needs to be carried out only once, in contrast with previous methods.This A·Bi staining technique permits excellent high contrast observation of glycogen particles in the cytoplasm of liver cells and myocardial muscle cells which could be seen to consist of fine granular substructures.

Comparative ultrastructural study on tris 1-aziridinyl phosphine oxide prefixed cuticles of some oligochaetes.

The cuticle of five species of Oligochaeta, chosen to represent differences in size and a variety of biotopes, was studied electron microscopically after fixation with the acrolein-TAPO-osmium tetroxide method. Five distinct layers in the cuticle of all studied species were found. Staining with lead and uranyl ions or with silver proteinate visualized basically the same structural components of the cuticle, but the degree of electron opacity and the distribution of the electron-opaque stain in these components differed according to the staining method used. Since the acrolein-TAPO-osmium tetroxide method visualized the cuticular zones preferentially stained by Thiéry's silver proteinate method, it was concluded that the TAPO method may be considered suitable for the visualization of polysaccharides. Staining with phosphotungstic acid provided some information on the composition of the cuticle of Oligochaeta not obtained by staining ultrathin sections with lead and uranyl ions nor with silver proteinate. The conclusion is that phosphotungstic acid binds to polysaccharides which do not contain vicglycol groups nor active sites responsible for the positive reaction with lead and uranyl salts. Structural components in the cuticle of the oligochaetes studied were characteristic for each species. The taxonomic value of such components, however, must be confirmed by examination of a larger number of species of oligochaetes.

Correlation between ultrastructure and histochemistry of lafora bodies.

Lafora bodies are composed of fibrillar and granular components in various concentrations. They are located in neuronal cell processes and perikarya and are frequently clearly intermingled with cytoplasmic elements. These components are electron-lucent, show a variable affinity for osmium, and are weakly stained by classical uranyl acetate stain following lead citrate. They exhibit a strong affinity for lead hydroxide and are periodic-acid reactive. They are not stained by the PTA technic for basic protein and/or acidic mucopolysaccharides. These results are in agreement with chemical studies according to which L. B are mostly composed of polyglucosans and suggest that both the granular and fibrillar ultrastructural components contain vic-glycol groups. The ultrastructure of L. B. is very similar to that of corpora amylacea, to deposits in basophilic degeneration of myocardium and in glycogenosis type IV.

Histochemistry of Mucosubstances in the Mantle of the Clam, Mercenaria mercenaria. II. MucoSubstances in the Second Marginal Fold

Histochemistry of Mucosubstances in the Mantle of the Clam, Mercenaria mercenaria. II. MucoSubstances in the Second Marginal Fold

The specific cytochemical demonstration in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups.

A technique is described for the localization in the electron microscope of periodate-reactive mucosubstances and polysaccharides containing vic-glycol groups. In this technique the sugar residues are oxidized by periodic acid and the resulting aldehydes condensed with pentafluorophenylhydrazine under specified conditions. Further increase in specific electron density is achieved by treating the hydrazone end-product with ammonium sulphide followed by osmium tetroxide to form an “osmium black”.

FORMAZAN REACTION AS A CONFIRMATORY TEST FOR ALDEHYDE GROUPS IN HISTOCHEMISTRY.

ONE of us has predicted elsewhere1 that not all neutral mucopolysaccharides can be demonstrated in tissue sections by the periodic acid–Schiff technique because the ‘dialdehydes’ which are formed by the periodate oxidation of their vic-glycol groups may be transformed into hemiacetals or hemialdals which are known to be unreactive towards Schiff's reagent in vitro2. On the other hand, all periodate-engendered ‘dialdehydes’ appear to react with aromatic hydrazines2,3 including salicyloyl hydrazide and 2-hydroxy-3-naphthoic acid hydrazide1. Therefore, in tissues which have been treated with periodic acid, those mucin sites which take up hydrazine reagents but not Schiff's solution probably contain ‘dialdehydes’ in the hemialdal and/or hemiacetal forms. This hypothesis has now been confirmed by showing that the formazan reaction is negative in these sites.