Some chemical, physical and histochemical properties of three diamine fractions obtained by gel chromatography from the high-iron diamine staining solution used for localizing sulphated mucosaccharides

Synopsis The coloured components in the high-iron diamine dye bath were separated into three fractions using column chromatography on Sephadex G-10. These fractions were called Fraction I, II and III in order of their emergence from the column. From atomic absorption measurements, part of Fraction I was found to be free of iron. Most of Fraction II and the whole of Fraction III contained only trace amounts of iron. Therefore, the three Fractions were investigated further. All experiments were carried out at pH 1.4 (corresponding to the pH of the original high-iron diamine bath).Fraction I was violet, Fraction II red-violet and Fraction III aniline-red; the extinction maxima in the visible region were 560, 526 and 540 nm respectively. On electrophoresis, the Fractions were not quite homogeneous, although most of Fractions I and II migrated in the same front and much faster than Fraction III. The high-iron diamine solution separated into two main fractions, one of which corresponded in colour and velocity to Fractions I and II and the other to Fraction III.In histochemical experiments, Fractions I, II and III bound to tissue sites containing sulphated mucosaccharides or nucleic acids; from histochemical enzyme digestion tests or by using purified materials in spot tests on cellulose acetate membrane, it was confirmed that the diamines were bound to RNA and DNA. However, when ferric chloride was added to any of the Fractions in an amount corresponding to that in the original high-iron diamine dye bath, the binding to tissue sites containing nucleic acids was inhibited but the reaction with sulphated mucosubstances was not affected. Also, in the presence of added ferric chloride, the anomalous binding of Fraction I to carboxyl groups of mouse sublingual gland sialomucin was prevented.It is concluded that ferric chloride in the high-iron diamine dye bath prevents diamine complexes from binding with nucleic acids, and apparently with carboxymucins too. Further, this conclusion substantiates our previous observations of the central role ferric chloride plays in making the histochemical high-iron diamine technique specific for sulphated mucosubstances.     

Investigations of dye binding in the sequential staining of mucosaccharides by Alcian Yellow-Alcian Blue. 1. Histochemical experiments on different animal and human mucosaccharides

Synopsis It may be assumed that, histochemically, carboxyl groups and sulphate half-ester groups of muco electrolyte concentration of the dye baths in the two steps of a sequential Alcian Yellow-Alcian Blue method. In the present study the specificity and reliability of this method has been investigated.When the staining conditions were the same in both steps, the second dye (Alcian Blue) was found to stain mucosubstances in spite of the prior staining with Alcian Yellow. Binding of Alcian Blue was observed in all but very dilute Alcian Blue solutions. The degree of Alcian Blue binding depended on the dye concentration and staining time of the second step (Alcian Blue), and it varied widely for different mucosubstances. Although an incomplete saturation of anionic groups with dye molecules in the first step cannot be completely excluded, it is thought that Alcian Blue displaces Alcian Yellow from the carboxyl and sulphate groups of mucosubstances in tissue sections.It seems that the sequential Alcian Yellow-Alcian Blue method, under the conditions investigated, does not provide a reliable means for differentiating carboxyl and sulphate groups of mucosbstances in tissue sections simultaneously, because the second dye obviously is capable of displacing the first dye from sulphate groups. However, it is possible to distinguish non-sulphated acid mucosaccharides from sulphated mucosaccharides.     

ULTRASTRUCTURAL LOCALIZATION OF ACID MUCOSUBSTANCES IN THE MOUSE COLON WITH IRON-CONTAINING STAINS

Selective ultrastructural staining of acid mucosubstances in sites containing histochemically identifiable sulfo- and sialomucins has been obtained in fixed cryostat sections with both ferric chloride and colloidal iron solutions. The rectosigmoid region of mouse colon was fixed in glutaraldehyde, formalin, or phosphate-buffered osmium tetroxide, and 40 µ cryostat sections of this material were treated with 0.1 to 0.4% ferric chloride or with a solution of dialyzed ferric chloride, ammonia, and glycerin. Specific staining depended upon the pH of the iron-containing solutions, and the optimal value was found to be approximately 2.0. Specific localization of acid mucosubstances has been noted in intracellular sites, including globules within colonic goblet cells and "deep crypt" mucous cells, small vesicles of the superficial nongoblet epithelial cells, and Golgi lamellae within each of these cell types. Extracellular material, presumed to be acid mucosubstance, was found on the surface of the epithelial microvilli and on the lumenal surface of capillary endothelium. Similar material formed a reticular network surrounding stromal cells, collagen bundles, and various colonic connective tissue elements.     

Tannic acid-metal salt sequences for light and electron microscopic localization of complex carbohydrates.

Use of tannic acid (TA), in sequence with ferric chloride, uranyl acetate or gold chloride resulted in staining of selective but sometimes different sites in paraffin sections. TA-uranyl acetate of TA-ferric chloride stained sites rich in complex carbohydrates, wherease TA-gold chloride stained the collagen of various connective tissues different shades of red-purple to gray-black. Applied to epoxy-embedded thin sections of tissues fixed with glutaraldehyde and not post-osmicated, TA-uranyl acetate and TA-ferric chloride imparted density to subcellular sites known to contain a high concentration of mucosubstances, such as secretory granules and cisternae of the Golgi complex of certain cells. TA-gold chloride proved unsatisfactory for ultracytochemistry because of its tendency to form globular precipitates on thin sections. The effect of blockage procedures at the light microscopic level indicated that vicinal glycols are not required for binding of TA to tissue sites. Electrostatic forces were shown to be of minimal significance, whereas hydrogen bonding appeared to play a part in both TA-tissue and TA-metal binding mechanisms.     

Histochemistry of mucosubstances in the mantle of fresh water mussel, Parreysia corrugata var. nagpoorensis (Lea) II. Mucosubstances of the middle marginal fold.

The results of the various histochemical reactions on mucosubstances indicate that in the middle fold of the mantle edge two types of mucus cells exist, one producing sulphomucins and the other neutral mucosubstances. The cells secreting neutral mucosubstances are few in number. The sulphated mucus is strongly alcianophilic. The alcianophilia persists when the tissues are stained with alcian blue in concentration up to 0-5 M magnesium chloride. Testicular hyaluronidase has no effect on the staining pattern of the mucus.     

Kurloff cell ultrastructure after combined formaldehyde-cetylpyridinium chloride fixation and high-iron diamine staining

Summary This study deals with the ultrastructure of the chondroitin sulphate proteoglycans of the Kurloff body, a large lysosomal organelle that stains metachromatically with Toluidine Blue and which is present in Kurloff cells (a blood cell unique to the guinea pig). Splenic tissues were fixed with 1% cetylpyridinium chloride (CPC) added to 4% paraformaldehyde and examined either after Spicer's high-iron diamine staining for sulphated anionic sites followed by post-fixation with ferrocyanide-osmium tetroxide or after a simple post-fixation with ferrocyanide-osmium tetroxide. CPC-precipitated sulphated sites were preferentially located at the periphery of the Kurloff body but, unexpectedly, were absent in the central matrix. Although their electron opacity was lower, these anionic sites were readily observable in the absence of HID-staining after sole post-fixation by ferrocyanide-reduced osmium. CPC-precipitated sulphated anionic sites were either associated with the myelin figures or constituted unexpected structures. They contained (i) tightly-stacked lamellae, with a very regular 4 nm periodicity, and (ii) groups of 2, 3, 4 short dense lines with a 3–5 nm periodicity. By taking into account the susceptibility of these HID-reactive structures towards chondroitinase ABC, these different sulphated components were assumed to be related to the proteochondroitin-4-sulphate previously characterized as the only major sulphated glycoconjugate of the Kurloff cell. Their colocalization with phospholipidic structures was suggested following, observation of sections treated by a chloroform-methanol mixture.     

Histochemical observations of the haemocytes of Locusta migratoria.

Five of the six categories of haemocytes of Locusta migratoria, that is, the plasmatocytes, spherule cells, granulocytes, coagulocytes and oenocytoids, contain conspicuous granules of mucosubstance in their cytoplasm. The mucosubstance has been characterized by using a series of histochemical tests, including Alcian Blue staining at different pH levels and salt concentrations, the periodic acid-Schiff (PAS) test, the high iron diamine test, enzymatic digestions and sequential staining methods. The results indicate that four different mucosubstances occur in a granular form, although not all four are found in every blood cell type. The mucosubstances are a neutral glycoprotein and neuraminidase-resistant, sulphated and non-sulphated sialomucins. The non-sulphated sialomucin occurs in both periodate-reactive and -unreactive forms.     

Histochemical studies of mucosubstances in the epidermis of a nonscaly teleost Mystus (Mystus) vittatus Bl. (Pisces-Bagridae)

The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.     

The detection by X-ray microanalysis of noradrenaline in sympathetic nerve terminals of the rat vas deferens

Summary The epidermis of Mystus (Mystus) vittatus contains two well differentiated mucous cells which secrete different mucosubstances. The goblet cells contain periodate reactive neutral mucosubstances, glycogen, testicular hyaluronidase resistant sulphated mucosubstances, and sialic acid rich glycoproteins. The clavate cells contain small amounts of neutral and sulphated mucosubstances and no glycoproteins. The difference in the histochemical nature of the two types of mucous cells is discussed in relation to their physiological activities.This investigation was supported by research Fellowship No. 7/176(138)77 from the Council of Scientific & Industrial Research, New Delhi to the first author     

Binding of Ferric Ions to Nuclei and Other Tissue Sites in Sections Stained for Sulfated Mucosubstances by the High-Iron Diamine Method

Binding of Fe³⁺ occurred in nuclei and several other sites when tissue sections, after a prior staining by the high-iron diamine (HID) method for sulfomucins, were immersed for 1 hr in 0.06 N HCl containing 1% potassium ferrocyanide (Prussian blue reaction). Apparently Fe³⁺, which is derived from FeCl₃ present in the HID dye bath, unites directly with these tissue components, although one cannot exclude the possibility that iron is first bound to colorless diamine complexes and then to tissues. The visualization of Fe³⁺ by ferrocyanide provides a simple way of obtaining a suitable nuclear stain combined with general counters taming for the HID method.     

Induced circular dichroism of the interaction between pinacyanol and algal alginates

The interaction between pinacyanol chloride and sodium alginate or guluronate-rich alginate is found to effect profound changes in the visible absorbance and circular dichroism spectra. Two different types of aggregates are observed depending on the relative dye/alginate concentrations. With a dye/alginate ratio at 1:1, a complex is deduced based on an analysis of Job’s method and conductometric titrations. Another complex forms at 1:10 dye/alginate ratio and only in the presence of alginate or guluronate-rich alginate. The two aggregates are in dynamic equilibrium according to the presence of isosbestic points in the visible spectra. The effects of pH and divalent cations on the spectra are studied. The 1:10 complex is damaged by addition of hydrochloric acid and divalent cations; however, at low concentration of these agents the spectra indicate conversion of the complex into the 1:1 aggregate. Models for the two complexes are proposed taking into account the preference of guluronate binding sites for chelating ions.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). I. Preparation and absorption properties in solution

Poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine) and poly(N^γ-acetoacetyl-L -diaminobutyric acid) form colored complexes with ferric ions in water/dioxane solutions. These complexes are soluble at pH values lower than 2.8 and show their maximum absorption at 257 nm in the uv and at 478 nm in the visible region; whereas the ferric complex of the model compound n-hexylacetoacetamide exhibits absorptions centered at 258 and 536 nm, respectively. It is shown that in the complex of the model compound one metal ion is bound per acetoacetamide group, while in the complexes of the three polymers two β-ketoamides side chains are bound per ferric ion under the same solvent, pH, concentration, and ionic strength conditions. The binding constants of ferric ions to the three polymers, and the formation constant of the ferric complex of the model compound are also evaluated.     

Chloride ion binding to human plasma albumin from chlorine-35 quadrupole relaxation.

The 35Cl nuclear magnetic quadrupole relaxation enhancement on binding of chloride ions to human plasma albumin (HPA) has been studied under conditions of variable temperature, pH, ionic strength, protein, and sodium dodecyl sulfate concentration. A small number (less than 10) of chloride ions, most of which are bound to the primary detergent binding sites, contribute a major portion of the relaxation enhancement (greater than 80% at neutral pH). A comparison of the pH dependence of the relaxation rate with the hydrogen ion titration curve, which was determined and analyzed, identified ten lysyl and arginyl residues as being involved in the chloride ion binding. These data, in conjuction with NaDodSO4 titrations at different pH values and the amino acid sequence of HPA, suggests that the high-affinity chloride-binding sites are doubly cationic at neutral pH. An irreversible dimerization at acidic pH and 5 x 10(-5) m HPA was detected. The data also indicate the presence of internal modes of motion in the expanded forms of the HPA molecule, probably an independent reorientation of domains. The rate of exchange of chloride ions was shown to be much higher than the corresponding intrinsic relaxation rate in the temperature range 2--26 degrees C and pH values ranging from 4.0 to 10.5. No indications of protein-protein interaction could be found up to the physiological concentration of ca. 6 x 10(-4)m HPA at either neutral or alkaline pH. The mechanistic basis for HPA's exceptional capacity for binding of inorganic anions was discussed.     

Cu2+ probe of metal-ion binding sites in melanin using electron paramagnetic resonance spectroscopy. I. Synthetic melanins

The isotope ⁶³Cu²⁺ has been used to probe the metal-ion binding sites of synthetic (autoxidized) catechol and 3,4-dihydroxyphenylalanine melanins using electron paramagnetic resonance spectroscopy. Samples were in aqueous media over a wide range of pH values. Assignments of the structures of the melanin-copper complexes are based in part on model studies of the complexes formed with melanin precursors, catechol and 3,4-dihydroxyphenylalanine, and with phenanthroline. Nearly all complexes involve just one or two ligands from melanin. In catechol melanin below pH 5.0, complexes with carboxyl groups are formed; above 6.0, Cu²⁺ forms complexes with phenolic hydroxyl groups. These same complexes were found in 3,4-dihydroxyphenylalanine melanin and binding of Cu²⁺ at amino acid type sites also was detected. After partial reduction of copper ions bound to 3,4-dihydroxyphenylalanine melanin, a weak signal of copper with four melanin ligands (oxygen and nitrogen in various combinations) was observed.     

Noncovalent interactions and coordination reactions in the systems consisting of copper(II) ions, aspartic acid and diamines

Interactions of aspartic acid between 1,3-diaminopropane (tn) and 1,4-diaminobutane (Put) in metal-free systems as well as in the systems including copper(II) ions were studied. The composition and overall stability constants of the complexes formed were determined by the potentiometric method. The interaction centres and coordination sites were identified by spectroscopic methods. Each of the ligands has both negative and positive interaction centres. In aspartic acid such centres are carboxyl groups and amine group, while in the polyamine molecules – protonated amine groups. The centres are also the potential sites of the coordination of metal ions. Analysis of the log K_e values of the adducts in the systems with polyamines has shown that the stability of the adducts in the metal-free systems depends on a significant degree on the steric factor that is the length of the polyamine. In some species the inversion effect, hitherto not reported in literature, was found. In the ternary systems including Cu(II) ions, only protonated species are formed, including molecular complexes with intermolecular interactions and metallation through the oxygen atoms of carboxyl groups and amine groups of the amino acid. In the adducts the protonated diamine is in the outer coordination sphere and is involved in noncovalent interactions with the anchoring CuH(Asp) or Cu(Asp) complexes.     

Release of diamine oxidase into plasma by glycosaminoglycans in rats

Plasma diamine oxidase (DAO) values are enhanced by intravenous injection of heparin which releases the enzyme, synthesized in small bowel enterocytes, from binding sites located on endothelial cells of the intestinal microvasculature. Intestinal DAO, in analogy with lipoprotein lipase (another heparin-released enzyme), is believed to be electrostatically linked to endothelial binding sites composed of a glycosaminoglycan (GAG) which is presumably heparan sulphate, but the complete mechanism of enzyme release is not known. In this study we assayed in rats the DAO-releasing capability of heparan sulphate, dermatan sulphate, chondroitin sulphate A and hyaluronic acid, all heparin related compounds. Heparan sulphate, a compound with the same hexosamine as heparin but with a lower concentration of sulphated iduronic acid, induced a very high release of DAO (3-fold less than heparin), while the other tested GAGs, composed of higher proportions of non sulphated uronic acid and with galactosamine instead of glucosamine, induced a significantly lower release. In rats treated with 60 mg heparan sulphate the significant decrease in ileal mucosal DAO activity indicates that, in analogy with heparin, the high plasma enzymatic activity induced is of enterocytic origin. It is suggested that the high charge density of the compounds tested, due to the degree of sulphatation, is the decisive factor in promoting the release of intestinal DAO.     

Histochemical reactions of gastrointestinal mucosubstances with high iron diamine after prior oxidation and methylation of tissue sections

Summary High iron diamine reactions after the prior methylation and oxidation of tissue sections with performic acid or potassium permanganate (metox-HID or ox-met-HID) in epithelial mucosubstances and in mucosal mast cells were studied in tissue samples from the human gastrointestinal tract and were compared with reactions with high iron diamine without any pretreatment (HID) and high iron diamine with the prior methylation (met-HID). High iron diamine reactions after the prior oxidation (met-ox-HID, ox-met-HID and ox-HID) demonstrated mucosubstances in a way which seemed to operate by the staining of acidic groups evoked by the oxidation of the tissue sections. These acidic groups were not blocked by the methylation. It was supposed that they are sulphonic acids resulting from sulphur groups (sulphydryls or disulphides) in some mucus glycoproteins. Met-ox-HID and ox-met-HID reactions seemed to stain mucosubstances and mast cells in a similar way but differed from the ox-HID reactions with the manner which could be interpretated to be due to the blocking of free sulphate ester groups in reactions of the former. Met-ox-HID (and ox-met-HID) positive mucosubstances were found in the foveolar epithelium of the stomach and in goblet cells of small and large bowel.The study was supported by grants from Sigrid Juselius Foundation and Paulo Foundation, Helsinki, Finland     

Morphological phenotypes and functional capabilities of submandibular parenchymal cells of the ferret investigated by protein, mucosubstance and enzyme histochemistry

Submandibular glands obtained post-mortem from mature ferrets of both sexes were examined with the use of light microscopical histochemical methods for proteins, mucosubstances and enzymes associated with cell functions or organelles. Demilunar cells showed carboxylated mucosubstances that were mainly non-sulphated, and diffuse activity for peroxidase, E600-sensitive esterase and acid phosphatase. Thiol groups were also detected in these cells. Central acinar cells showed sulphated mucosubstances, disulphides and reticular staining for thiamine pyrophosphatase. Intercalary ducts showed diffuse activity for NADH and NAD(P)H dehydrogenases. Striated ducts contained protein, tryptophan, disulphides, neutral mucosubstances and E600-sensitive esterase periluminally. Basally, the striated ductal cells showed variable activity for peroxidase, cytochrome oxidase, succinate dehydrogenase and acid phosphatase. Basolateral plasma membranes of these cells exhibited ouabain-sensitive Na,K-ATPase activity. The collecting ducts were characterized by variable periluminal staining for acid phosphatase, -glucuronidase, acid -galactosidase and E600-resistant esterase. The results suggest that the histological appearances of the acini of the submandibular gland of the ferret are dependent on the synthesis of secretory acid glycoproteins, that the striated ducts are involved with the secretion of tryptophan-rich product comprising neutral glycoproteins and showing esterase activity and with marked transport of ions and that the collecting ducts are involved with absorption.     

Insect mucosubstances. I. The mucosubstances of developing connective tissue in the locust,Locusta migratoria

Synopsis A layer of collagenous connective tissue develops around the ejaculatory duct of the male locust,Locusta migratoria, during the fourth and fifth instars and the first ten days of the adult stage. The mucosubstances associated with this tissue have been characterized by using a series of histochemical reactions, including Alcian Blue staining at different pH levels and salt concentrations the periodic acid-Schiff test and recent modifications of it, the high iron diamine test and enzymatic digestions. The results indicate that sulphated glycosaminoglycans accumulate during the developmental period, so that in the mature adult, the connective tissue probably contains chondroitin and dermatan sulphates, and possibly some keratan sulphate. Neutral glycoproteins also occur in the tissue.     

A nitrous acid procedure as a selective histochemical means of eliminating the N-sulphates of glycoconjugates

Summary A nitrous acid procedure has been shown to lead to the elimination of N-sulphates in sections of a series of tissues containing sulphated glycoconjugates. Two groups of sulphated glycoconjugate-containing tissues were used; one contained N-sulphates and other was devoid of such groupings. In the first group of tissues, mast cells of different origins and renal glomeruli in the rat were employed. Xiphoid and tracheal cartilage matrix, submandibular and sublingual gland acini and gastric, duodenal and colonic mucosae were used in the second group. Sections were treated with nitrous acid and then stained with Alcian Blue pH 1.0, high iron diamine or Aldehyde Fuchsin for sulphated glycoconjugates. Such treatment was found to diminish the staining intensities exclusively in N-sulphated glycoconjugate-containing structures such as mast cell granules and renal glomerular basement membrane, providing a means of chemically eliminating N-sulphates of glycoconjugates in tissues.