Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects

Summary Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1m MgCl₂, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacked purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl₂ staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl₂ were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.     

Metachromatic staining and electron dense reaction of glycosaminoglycans by means of Cuprolinic Blue

Summary The cationic phthalocyanin-like dye Cuprolinic Blue, unlike phthalocyanin dyes such as Alcian Blue or Astra Blue, can definitely exhibit a clear metachromatic reaction with appropriate substrates, The application of Cuprolinic Blue to epoxy-embedded semithin sections revealed that mast cell cytoplasmic granules, goblet cell mucin and cartilage matrix stained in violet shades (metachromatic), whereas nuclear chromatin presented a bright blue coloration (orthochromatic). The metachromatic structures showed a high degree of contrast when ultrathin sections treated with Cuprolinic Blue were examined by electron microscopy.Cytophotometric measurements of stained components from the large intestine showed different absorption maxima: at 580 nm for mucin and at 640 nm for nuclei. The spectroscopical analysis revealed a clear-cut metachromatic shift when the dye was in the presence of chondroitin—4-sulphate. The addition of aluminium metal to Cuprolinic Blue solutions resulted in a striking spectral change; under such conditions the dye showed absorption maximum at 530 nm.     

Staining of proteoglycans in mouse lung alveoli. I. Ultrastructural localization of anionic sites

Summary In order to contrast anionic sites, in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules.After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl₂ concentration used during staining. At 0.4m MgCl₂, the length was mostly within the range 100–180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.     

Differential staining of glycosaminoglycans in the predentine and dentine of rat incisor using Cuprolinic Blue at various magnesium chloride concentrations

Summary Rat incisors were fixed with a solution of 0.05% Cuprolinic Blue and 2.5% glutaraldehyde in the presence of various concentrations of MgCl₂ according to the critical electrolyte concentration (CEC) principle. This method allows glycosaminoglycans (GAG) to be properly preserved and visualized. Small granules were stained by the cationic dye in the predentine in the absence of MgCl₂. These granules grew in size and became more electron-dense when the concentration of the electrolyte was increased. Larger ribbon-like structures and granules were seen when 0.3M MgCl₂ was used. In the dentine, tiny dots in close association with the surface of the collagen fibres, or their periodic striations, were positively stained. A thick electron-dense band located on the dentine side at the predentine-dentine junction was seen both with and without 0.05 M MgCl₂. With higher concentrations of the electrolyte (0.1–0.3 M), this band was reduced to a very thin line located at the border of the dentine, along with mineralizing collagen fibres. This demonstrated the presence of GAG at the dentine surface and therefore indicated that GAG may play a role as nucleator agent.     

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.     

Antibody-dependent cellular cytotoxicity in the guinea pig: The role of the Kurloff cell

In the guinea pig, the killer cell in in vitro ADCC assays, is found to be the Fc receptor-bearing Kurloff cell. This killer Kurloff cell is under hormonal control, estrogen treatment significantly increasing the Kurloff cell numbers in blood, spleen, and thymus, and markedly augmenting the lytic capacity of these lymphoid compartments. The cytotoxic killer lymphocyte, if present, appears to be a minor effector cell in guinea pig ADCC. Selective lymphocyte subpopulation and Kurloff-cell depletion procedures reveal that the killer Kurloff cell may also possess a variety of other membrane markers (T⁺, C3⁺, Ig⁺). The particular membrane profile of a cytotoxic Kurloff cell is determined by its lymphoid site of residence and the hormonal status of the animal.     

Histochemical evidence of a functional heterogeneity of the chondrocytes of adult canine articular cartilage

Synopsis Twenty humeral heads were collected from 10 adult English Pointer dogs, fixed in 15% formalin containing cetylpyridinium chloride, decalcified, processed for paraffin sections, and cut serially. The articular cartilage was studied by staining principally with Alcian Blue in the presence of 0.4 or 0.9m MgCl₂ with and without a van Gieson counterstain. The results of the differential staining procedures demonstrated the existence of two groups of chondrocytes with distinctly different staining affinities. One group reacted intensely for the presence of protein-polysaccharides within its cytoplasm while the other group completely lacked this property. An approximate proportionality of 31 of the protein polysaccharide-positive and-negative chondrocytes was observed in the tangential layer and upper intermediate zone. In the lower intermediate zone, radiate zone, and zone of calcified cartilage, the chondrocyte types were present in equal proportions. Staining with Alcian Blue in the presence of 0.9m MgCl₂ with and without a van Gieson counterstain indicated a further subdivision of the protein-polysaccharide positive group of chondrocytes. This blocking technique has been reported to distinguish between chondroitin sulphate and high mol. wt. keratosulphate. Thus, based upon a greatly decreased number of the blue-stained chondrocytes after staining with Alcian Blue in the presence of 0.9m MgCl₂ the hypothesis is put forward that some chondrocytes produce primarily chondroitin suphate and others produce both chondroitin sulphate and keratosulphate.     

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.     

Natural killer activity of kurloff cells: A direct demonstration on purified kurloff cell suspensions

In order to study their natural killer effect, guinea pig splenic Kurloff cells were fractionated by Percoll discontinuous density gradient centrifugation. Kurloff cells were collected and tested for cytotoxicity in a 24-hr chromium-release test. Comparison of different splenic cellular samples (of males or estrogenized females) with increasing percentage of Kurloff cells, revealed a highly significant positive correlation (r = 0.93, α < 0.01) with the cellular cytotoxicity developed against the K 562 target cells.     

Histochemical demonstration of hyaluronic acid molecules by Alcian Blue

Summary Specimens of vitreous humour (monkey eye), Wharton jelly (human umbilical cord) and commercial hyaluronates were immersed in buffered fixative solutions containing either aldehydes and Alcian Blue, or aldehydes and Alcian Blue with MgCl₂ as electrolyte. Two MgCl₂ concentrations were used, 0.025m and 0.3m. Immersion in both solutions induced formation of precipitates which were postfixed in OsO₄, dehydrated and embedded for thin section electron microscopy. The use of the same fixative solution produced morphologically comparable precipitates from all three materials. The precipitates, especially after fixation in the presence of electrolyte, were composed of linear, unbranched filaments, frequently aggregated into bundles. The filaments were considered to be molecules of hyaluronic acid.Part of this work was presented at the 10th Meeting of the European Club for Ophthalmic Fine Structure, Copenhagen, September 3–4, 1982.     

Ultrastructure of proteoglycans in the specific granules of guinea-pig basophilic leukocytes as demonstrated by cuprolinic blue staining

The ultrastructure of sulphate proteoglycans in basophil granules was examined using cytochemical procedures designed to stabilize and visualize these highly anionic macromolecules in situ. Unfixed or glutaraldehyde-prefixed guinea-pig spleen cells were submitted to fixation/staining in 2.5% glutaraldehyde, 0.2% cuprolinic blue (CB; a cationic phthalocyanin dye) and 0.2 or 0.3M MgCl₂ with or without glycosidase treatments. Abundant electron-dense precipitates were present throughout the granule matrix. The stained structures were often arranged in a quasi-crystalline typical banded pattern. Negative control basophils had no electrondense precipitates. Digestion with chondroitinase ABC destroyed the CB-positive electron-dense banded or filamentous patterns while sialidase treatment did not, but led to larger CB-positive filaments in the cytoplasm near the granules. Taking into account their high anionicity, as shown by the stability of dye binding in the presence of 0.3M MgCl₂, and their susceptibility to chondroitinase ABC, the CB-precipitates are assumed to be related to the sulphated proteoglycans previously characterized in basophil granules. The CB-positive crystalline or filamentous network of the granule matrix is also assumed to reflect the in situ location and organization of these intracellular proteoglycans and may be involved in maintaining the shape of the granule.     

Fibrinogen or its derivatives in the Kurloff cells.

We have investigated the occurrence of fibrinogen or its derivatives in the Kurloff bodies of female guinea pigs by means of immunofluorescence, histochemistry and ultrastructural analysis. The Kurloff bodies are fluorescent after incubation with anti-guinea pig fibrinogen immuno-globulins coupled with fluorescein isothiocyanate. PTAH, Martius scarlet blue, Masson 44--41, Picro-Mallory V stain the Kurloff bodies just as they stain fibrin and fibrinoid. However, the ultrastructure of the central mass of the Kurloff body is finely granular without fibrils. Thus the Kurloff body contains no fibrin but incompletely polymerized fibrinogen derivatives or breakdown products of fibrin.     

Experimental mucopolysaccharidosis: Preservation and ultrastructural visualization of intralysosomal glycosaminoglycans by use of the cationic dyes cuprolinic blue and toluidine blue

Summary The cationic dyes Cuprolinic Blue (CB) and Toluidine Blue (TB) were used to preserve the intralysosomal storage material accumulating in tilorone-induced mucopolysaccharidosis. As shown in previous studies, the stored glycosaminoglycans (GAGs) are leached during the conventional fixation procedure, with the result that the lysosomes appear empty. In the present study, the liver, spleen, and cornea-conjunctiva of tilorone-treated rats were examined. The application of CB in the presence of 0.1 M or 0.3 M MgCl₂ simultaneously with, or subsequently to the primary fixative yielded electron-dense precipitates within the storage lysosomes. When TB (0.1%) was added to the primary fixative, the storage lysosomes contained filamentous structures arranged in reticular patterns. With increasing TB concentrations (up to 1%) the lysosomes increasingly often showed apparently amorphous storage material which was continuous with the reticular filamentous structures. Similar ultrastructural patterns were obtained with GAG-TB complexes prepared in vitro. The intralysosomal storage material preserved by TB is interpreted as GAG-TB precipitates. In conclusion, the use of CB provides a method which allows direct cytochemical demonstration of the subcellular sites of GAG-storage. The use of TB represents an easy method to obtain electron micrographs pathognomonic of the mucopolysaccharidosis induced by tilorone and congeners. Either method may be helpful to detect this adverse drug effect at the subcellular level.     

The characterization of a protein–polysaccharide isolated from Kurloff cells of the guinea pig

Kurloff cells of guinea pigs increase in number and accumulate in the spleen on oestrogen treatment. Because they contain metachromatic inclusions and are considered to be lymphocytes they were examined as a possible model for mucopolysaccharidoses like Hurler's syndrome, where some lymphocytes are also metachromatic. Oestrogen treatment produced a large increase in a glycosaminoglycan resembling chondroitin 4-sulphate in chemical analysis, chromatographic behaviour and i.r. spectrum but with an additional strong band at 805cm(-1). Material isolated without proteolysis behaved on gel chromatography as a multiple-chain protein-polysaccharide whose molecular size was decreased by proteolysis. It contained xylose and galactose in molar proportions with serine, compatible with the presence of the same linkage region as in cartilage chondroitin 4-sulphate proteins and which likewise underwent alkaline beta-elimination. Kurloff glycosaminoglycan chains were significantly longer than chondroitin sulphate chains of cartilage protein-polysaccharides as assessed by gel chromatography and the molar ratios of galactosamine to xylose or to serine. Kurloff cells thus contain intact rather than partially degraded protein-polysaccharide and hence are not analogous to Hurler cells, and their electron micrographs were also different. The purified Kurloff protein-polysaccharide and glycosaminoglycan isolated here has been shown by Marshall, Swettenham, Vernon-Roberts & Revell (1970) to be toxic specifically to macrophages at extremely low concentrations in vitro, unlike chondroitin sulphate of protein-polysaccharides from cartilage. The toxic constituent may account for the i.r.-absorption band at 805cm(-1). Although active incorporation of [(35)S]sulphate occurs at early stages of Kurloff-cell induction (Marshall et al. 1970), the fully developed Kurloff cell studied here showed very low incorporation in vitro and in vivo, suggesting that the inclusions are specialized for the storage of the toxic material.     

Stimulation of gallbladder fluid and electrolyte absorption by butyrate

Summary Gallbladder fluid and electrolyte transport was investigatedin vitro. In guinea pig gallbladder, equimolar substitution of acetate, propionate, butyrate or valerate for HCO₃ was increasingly effective in stimulating fluid absorption. The stimulatory potency of these compounds was a function of their chloroform water partition coefficients. The stimulatory effects of the isomers isobutyrate and isovalerate were less than predicted from their partition coefficients. Acidification of the gallbladder lumen, however, was strictly dependent on the partition coefficients for all of the above fatty acids. Unidirectional²²Na fluxes were measured in rabbit and guinea pig gallbladders under short-circuit conditions. In the presence of butyrate stimulation of net Na flux was due entirely to an increase in the mucosal-to-serosal Na flux. Stimulation by butyrate was abolished by its omission from the mucosal bathing solution. The transepithelial electrical potential difference in both rabbit and guinea pig gallbladder became more lumen positive following mucosal but not serosal addition of butyrate. Net¹⁴C-butyrate fluxes were too small to account for stimulation of Na absorption in either species. Butyrate stimulation of Na absorption by guinea pig gallbladder was abolished by increasing the bathing pH from 7.4 to 8.1. Tris buffer (25mm) partially inhibited butyrate-dependent gallbladder fluid absorption by rabbit and guinea pig at pH 6.4 and 7.0, respectively, and completely at pH 8.4. These results reveal a marked similarity between butyrate and HCO₃ stimulation of gallbladder NaCl and fluid absorption. The results are best explained by a double ion-exchange model, in which butyrate (HCO₃) in the mucosal solution acts to maintain the intracellular supply of H⁺ and butyrate (HCO₃) for countertransport of Na and Cl, respectively.     

Isolation of Kurloff cells by Percoll density gradient centrifugation. Protein labeling with 35S-methionine of these cells

Large populations of splenic Kurloff (150 - 200 X 10(6) Kurloff cells) were obtained from estrogenized guinea pigs by isopycnic centrifugation in a Percoll solution of 1.085 g/ml starting density. The Kurloff cells settled at a buoyant density of about 1.100 g/ml. The purity of these cell suspensions reached 95%, as assessed by phase contrast microscopy and by specific staining. The viability assessed by Trypan blue exclusion test was also about 95%. Moreover, the good transmission electron microscopic appearance of these Kurloff cells and their ability to take up 35S-methionine in culture confirmed their physiological integrity. By autohistoradiography, this protein labeling was localized between the nucleus and the Kurloff body, and also on the Kurloff body itself. This data reinforces the hypothesis of de novo synthesis of the Kurloff body.     

Phenotypic expression of proteoglycan in mast cells of the human nasal mucosa

Summary The phenotypic expression of the proteoglycan of human mast cells in the nasal mucosa and normal skin was analysed using histochemical techniques. Nasal mucosa was obtained from normal subjects, from patients with seasonal allergic rhinitis before and during the pollen season and from patients with nasal polyps. In the latter groups, specimens were taken from both polyp tissue and adjacent nasal mucosa. Formaldehyde treatment blocked the cationic dye binding in 75–84% of the mast cells located in the nasal mucosa, as compared to the optimum fixation with IFAA (iso-osmotic formaldehyde-acetic acid). A significantly lower degree of blocking of dye binding was obtained in the human skin where 45% of the mast cells were susceptible to formaldehyde treatment (P<0.01). The mast cells of the polyp tissue also showed a relatively low degree of blocking (54%), which was significantly lower than the blocking of mast cells of the nasal mucosa taken from the same individuals (P<0.05). Staining of serial tissue sections in Alcian Blue containing graded concentrations of MgCl₂ was used to determine the critical electrolyte concentration (CEC) of the dye binding, defined as the salt concentration at which the staining of 50% of the mast cells is extinguished. The CEC of the skin mast cells was 0.64m MgCl₂ which is significantly higher than that of the mast cells of the nasal mucosa of normal subjects [0.49m (P<0.05)], allergic subjects [0.52m (P<0.01)], patients with polyp disease [0.52m (P<0.01)] and the polyp tissue proper [0.57m (P<0.05)]. This implies that mast cells of the nasal mucosa contain glycosaminoglycans of a relatively lower charge density and/or molecular size than the connective tissue mast cells found in the human skin. A similar difference has been observed between rat mucosal mast cells, containing a chondroitin suphate proteoglycan, and rat connective tissue mast cells which contain a heparin proteoglycan. However, unlike the rat mucosal cells, the mast cells of the human nasal mucosa showed a weakly fluorescent Berberine binding and, like the rat connective tissue mast cells, entirely lost the ability to bind Toluidine Blue after treatment with nitrous acid. Such treatment results in a deaminative cleavage of heparin and heparan sulphate, but does not degrade chondroitin sulphate. These results provide further evidence of the existence of a distinctive mucosal mast cell phenotype also in man. It is suggested that the lower CEC of the mucosal mast cells is an expression of a content of haparan sulphate, while the relatively higher CEC of the skin mast cells is compatible with a content of heparin.     

Staining of proteoglycans in mouse lung alveoli. II. Characterization of the Cuprolinic Blue-positive, anionic sites

Summary The nature of Cuprolinic Blue-positive anionic filaments in mouse lung alveoli has been characterized. The contrast of filaments in the alveolar basement membrane of type I epithelial cells was lost on treatment with nitrous acid and pronase (without prefixation). In contrast, neither neuraminidase, chondroitinase ABC or AC, norStreptomyces hyaluronidase had any effect. Treatment with pronase (after prefixation) and 2.0m MgCl₂ (after prefixation) also had no effect, indicating that the filaments are heparan sulphate proteoglycans. The filaments in the alveolar basement membrane of type II epithelial cells and in the capillary basement membrane of the endothelial cells were also nitrous acid sensitive, but chondroitinase ABC-insensitive. A model in which the whole alveolus contains a single layer of heparan sulphate-containing proteoglycan monomers is proposed. Furthermore, the collagen fibril associated filaments remained unaffected after treatment with nitrous acid, neuraminidase orStreptomyces hyaluronidase, or after digestion with pronase (after prefixation) and treatment with 2.0m MgCl₂ (after prefixation). These filaments, however, could no longer be detected when digestion with chondroitinase ABC or pronase (without prefixation) was applied; chondroitinase AC treatment clearly affected the filaments, although they still were visible. These results indicate that the filaments are dermatan sulphate-containing proteoglycans. Some functional aspects of the proteoglycans are discussed.     

Ultrastructural localization of a chondroitinase-sensitive, cuprolinic blue-positive filament in developing mouse lung

By use of the cationic dye Cuprolinic Blue in a critical electrolyte concentration method, the lungs of mice ranging from the late fetal stage (17 days of gestation) to the puberal stage (27 days) were surveyed for their proteoglycans. A large Cuprolinic Blue-positive filament is present within the connective tissue of lungs of late fetal and young postnatal mice. It is mostly located at the boundary between large extracellular matrix structures and electron microscopically empty areas, but sometimes also at the surface of fibroblast-like cells. The stainability of the filament disappears after treatment with chondroitinase ABC or chondroitinase AC, but not after treatment with nitrous acid. The Cuprolinic Blue-positive structure appears to be most abundant around 2 days postnatally. From day 10 on, its number decreases dramatically, and it can be no longer observed in the lungs of 27-day-old mice.     

Studies on the permeability of living cells

Summary The penetration of the dye, dahlia, into the sap ofNitella has been determined in the presence of NaCl, KCl, MgCl₂ and CaCl₂ at various concentrations.NaCl is the least effective and MgCl₂ was the most effective in preventing penetration of the dye.It was found that NaCl antagonizes the action of CaCl₂ in certain proportions to a small degree but not sufficiently to permit the dye to penetrate at a normal rate.Published by permission of the Surgeon General.