Ruthenium red binding to cell coat in neoplastic neurogenic cell lines in culture.

The cell coat of cultivated fetal rat brain cells as well as malignant rat neurogenic cell lines in culture were studied by transmission electron microscopy with the ruthenium red staining technique. Some of the transformed cell lines demonstrated alteration in the bindng properties of ruthenium red to the cell surface. Otherwise no significant correlation between the visualized cell coat thickness and neoplastic transformation was noted.     

A SPECIFIC HYDROXYLAMINE-FERRIC CHLORIDE REACTION FOR HISTOCHEMICAL LOCALIZATION OF PECTIN

An alkaline solution of hydroxylamine is caused to react with the methyl esters of pectin present in sections of plant tissues to produce hydroxamic acids which, after acidification with hydrochloric acid, yield a red color upon addition of 2.5% ferric chloride solution. The test is specific and preferable to the use of ruthenium red for localization of pectin in the compound middle lamella. Subjecting sections to methylation and using reagents made up in 60% alcohol increases the intensity and sharpness of the color formation.     

Dietary fiber inhibits the incidence of hepatic metastasis with the anti-oxidant activity and portal scavenging functions

Dietary fiber is described as the proportion of plant foods not digested in the human small intestine. Among the various kinds of pectin, apple pectin exerts a bacteriostatic action and therefore may change the composition of the intestinal flora. The diet supplemented with 20% apple pectin significantly decreased the number and the incidence of AOM-induced colon tumors in rats. The prostaglandin E2 (PGE2) level in the distal colonic mucosa and blood of portal vein was lower in rats fed 20% pectin than those fed the basal diet. The ability of apple pectin to decrease PGE2 was dose-dependent and those results suggest an anti-inflammatory effect in the bowel. Rats fed apple pectin showed a significantly lower incidence of hepatic metastasis than those fed the basal diet. To determine whether the anti-inflammatory effect of Lactobacillus on hepatic metastasis model same as apple pectin, Lactobacillus casei was selected. Metastatic nodules were significantly reduced, especially in the group receiving pretreatment. Apple pectic oligosaccharides with small molecular weights showed highly inhibitory effects on hypoxanthin-xanthin oxidase and ferrous sulfate-hydrogen peroxide. The scavenging activity of apple pectic oligosaccharides was suitable and accelerated at high temperatures (120 degrees C, 30 min.). Apple pectin and Lactobacillus have a scavenger effect in the intestinal digestion and portal circulation system as an anti-inflammatory food and have very important function for the prevention of hepatic metastasis.     

Block by ruthenium red of the ryanodine-activated calcium release channel of skeletal muscle

The effects of ruthenium red and the related compounds tetraamine palladium (4APd) and tetraamine platinum (4APt) were studied on the ryanodine activated Ca2+ release channel reconstituted in planar bilayers with the immunoaffinity purified ryanodine receptor. Ruthenium red, applied at submicromolar concentrations to the myoplasmic side (cis), induced an all-or-none flickery block of the ryanodine activated channel. The blocking effect was strongly voltage dependent, as large positive potentials that favored the movement of ruthenium red into the channel conduction pore produced stronger block. The half dissociation constants (Kd) for ruthenium red block of the 500 pS channel were 0.22, 0.38, and 0.62 microM, at +100, +80, and +60 mV, respectively. Multiple ruthenium red molecules seemed to be involved in the inhibition, because a Hill coefficient of close to 2 was obtained from the dose response curve. The half dissociation constant of ruthenium red block of the lower conductance state of the ryanodine activated channel (250 pS) was higher (Kd = 0.82 microM at +100 mV), while the Hill coefficient remained approximately the same (nH = 2.7). Ruthenium red block of the channel was highly asymmetric, as trans ruthenium red produced a different blocking effect. The blocking and unblocking events (induced by cis ruthenium red) can be resolved at the single channel level at a cutoff frequency of 2 kHz. The closing rate of the channel in the presence of ruthenium red increased linearly with ruthenium red concentration, and the unblocking rate of the channel was independent of ruthenium red concentrations. This suggests that ruthenium red block of the channel occurred via a simple blocking mechanism. The on-rate of ruthenium red binding to the channel was 1.32 x 10(9) M-1 s-1, and the off-rate of ruthenium red binding was 0.75 x 10(3) s-1 at +60 mV, in the presence of 200 nM ryanodine. The two related compounds, 4APd and 4APt, blocked the channel in a similar way to that of ruthenium red. These compounds inhibited the open channel with lower affinities (Kd = 170 microM, 4APd; Kd = 656 microM, 4APt), and had Hill coefficients of close to 1. The results suggest that ruthenium red block of the ryanodine receptor is due to binding to multiple sites located in the conduction pore of the channel.     

Use of colloidal gold and ruthenium red in stereo high-voltage electron microscopic study of Con A-binding sites in mouse macrophages

Concanavalin A (Con A)-binding sites were labeled with colloidal gold (CG), stained with ruthenium red, and observed under a high-voltage electron microscope. Mouse peritoneal macrophages were labeled by the indirect Con A/CG labeling method at 0 degree C. After washing, some of the cells were incubated in phosphate-buffered saline (PBS) at 37 degrees C. The specimens were then stained with ruthenium red, to enhance the contrast of the cell surface, and embedded in Epon. Sections (0.3 approximately 3 micron thick) were cut and examined by high-voltage electron microscopy at accelerating voltages of 200 approximately 1,000 kV. Staining with ruthenium red provided a strong contrast of the cell surface and the invaginating tubules beneath it against the cytoplasm; in thick sections, both of them were clearly seen by stereomicroscopy. CG particles which represented Con A-binding sites were also sufficiently electron dense to be recognized by high-voltage electron microscopy of thick sections. The two- and three-dimensional distribution of CG particles on the ruthenium-red-positive cell surface was clearly visualized. At 0 degree C, Con A-binding sites were randomly distributed on the cell surface. The redistribution and endocytosis of Con A-binding sites were seen at 37 degrees C. The three-dimensional organization of membrane invagination, which represented the process of endocytosis, was clearly seen by stereomicroscopy. The combination of CG labeling and ruthenium red staining is a useful method for high-voltage electron microscopic analysis of the two- and three-dimensional distribution of CG-labeled ligands on the cell surface in thick sections.     

Adriamycin Binds to the Matrix of Secretory Granules during Mast Cell Exocytosis

The antineoplastic drug adriamycin induces exocytosis in rat peritoneal mast cells followed by a significant uptake of the drug into the secretory granules. The drug is fluorescent, allowing visualization of its accumulation and binding to mast cell granules by fluorescence microscopy. At the same time, the well known inorganic dye ruthenium red was used as a probe because of its great affinity for heparin in the mast cell secretory granules as visualized by bright field microscopy. Competition between adriamycin and ruthenium red for binding to the negatively charged matrix of granules was demonstrated. Biochemical studies were also performed to confirm microscopic observations. Adriamycin may be of interest for studying mast cell secretion; it is not only a strong fluorescent dye for mast cell granules that are in communication with the extracellular space, but it also induces mast cell exocytosis.     

Electronmicroscopic histochemical investigation of the surface of the cell membrane of the renal tubules with the use of ruthenium red and lantanum nitrate.

The renal tubules were investigated with the use of ruthenium red and lanthanum nitrate to titrate the carbohydrate groups which are localized on the differentiated surface of the cell membranes and intercellular spaces. Ruthenium red visualized the surface coat which is tightly bound to the outer lamellae of the cell membrane. Lanthanum nitrate used in this investigations is a valuable marker of the intercellular spaces. The applied markers have visualized in the kidney the canals which are originated from the foldings of membranes of the tubular cells which adhere to the basal membrane. The markers used in combination with the fixative glutaraldehyde-paraformaldehyde give an electrondense envelope of the cell membrane which is more distinct as compared with specimen treated with glutaraldehyde and markers only.     

Improved Staining of Extracellular Polymer for Electron Microscopy: Examination of Azotobacter, Zoogloea, Leuconostoc, and Bacillus

Phase contrast, ultraviolet microscopy, and freeze-etching were used to determine the amount of exocellular polymer surrounding unfixed cells of four genera of bacteria: Azotobacter vinelandii, Zoogloea ramigera, Leuconostoc mesenteroides, and an acid-tolerant, floc-forming Bacillus species. Thin-sectional electron microscopy was employed to measure the effectiveness of a modified ruthenium red staining method. The results obtained with this modification of ruthenium red staining technique were compared to results obtained when previously proposed ruthenium red methods of fixation were employed. The results of these relations were then compared to the amounts of exocellular material as determined with phase-contrast microscopy, ultraviolet microscopy, and freeze-etching. The data obtained indicate that improved fixation of exocellular polymer is achieved when cells are pretreated with ruthenium red as described herein. In addition, the modified methods also reveal cytological detail not apparent when other methods of ruthenium fixation are employed.     

Modification of bursting in a Helix neuron by drugs influencing intracellular regulation of calcium level

The effect of ruthenium red, caffein and EGTA (ethyleneglycol tetraacetic acid) influencing intracellular Ca2+ level as well as that of pH-lowering was investigated on identified RPal neuron of Helix pomatia characterized by bimodal pacemaker (bursting) activity. Drugs were applied both extracellularly and intracellularly. Intracellular injection was performed from micropipettes by pressure. It was found that intracellular injection of ruthenium red, caffein, EGTA and pH-lowering caused immediate short hyperpolarization and suspension of bursting. The effect of caffein and lowering of pH was biphasic, hyperpolarization was followed by an increase of spiking. Following EGTA injection the amplitudes of interburst hyperpolarizing waves decreased, and prolongation of spikes occurred. Extracellular application of ruthenium red caused slight depolarization, while caffein produced mainly effects that were similar to those of the intracellular injection. Adding EGTA into the bath resulted in cessation of bursting, and later on also spike generation was blocked. All these effects could be eliminated by washing. It is concluded that Ca-influx during spiking cannot be considered as a single factor in maintaining bursting activity, nevertheless, intracellular binding and liberation of Ca depending on the cell metabolism should also be taken into consideration as a possible mechanism of burst regulation.     

Cytochemistry and morphology of synaptic structures in the cerebral cortex of rat.

Studies have been carried out on the synapses in the cerebral cortex of rat by using impregnation with ethanolic solution of phosphotungstic acid, contrast staining with ruthenium red and impregnation with bismuth iodide, with or without subsequent uranyl acetate and lead citrate staining. It has been established that dense projections are adequately visualized with methods demonstrating basic chemical groups (phosphotungstic acid and bismuth iodide), whereas the synaptic vesicles are stained by techniques demonstrating acid chemical groups (ruthenium red and uranyl acetate and lead citrate). On the basis of these observations a hypothesis is forwarded concerning the mechanisms of migration of synaptic vesicles towards the presynaptic membrane. Measurements of the parameters of the dense projections suggest that the configuration of the presynaptic vesicular grid is not uniform along the presynaptic areas.     

THE STRUCTURE OF THE ROOT NODULES OF CEANOTHUS SANGUINEUS AND CEANOTHUS VELUTINUS,WITH SPECIAL REFERENCE TO THE ENDOPHYTE

Furman , Thomas E. (Escuela Agrícola Panamericana, Tegucigalpa, Honduras.) The structure of the root nodules of Ceanothus sanguineus and Ceanothus velutinus, with special reference to the endophyte. Amer. Jour. Bot. 46(10): 698–703. Illus. 1959.—Past studies of non-leguminous root nodules have cited the endophytes of these structures as bacteria, filamentous fungi, actinomycetes, and members of the Plasmodiophorales. In this study, nodules of Ceanothus sanguineus and of Ceanothus velutinus were examined by using a variety of fixatives and staining methods as checks against artifacts, as well as histochemical tests and phase-contrast microscopy of living material. It was concluded that the nodules of these species were modified short roots, not homologous with leguminous nodules, inhabited by a filamentous microorganism which resembled most closely members of the described genus Streptomyces. The thallus of the endophyte had a definite, firm wall structure which absorbed ruthenium red stain actively, thus indicating a high pectin content.     

Fungal isolates from natural pectic substrates for polygalacturonase and multienzyme production

Pectin rich wastes and waste dump yard soils were screened and eighty pectinolytic fungal isolates were obtained by enrichment culturing and ruthenium red plate assay. Eight isolates with higher zones of pectin hydrolysis were selected and tested for polygalacturonase production. One isolate identified as Aspergillus awamori MTCC 9166 with highest polygalacturonase activity was tested for utilization of raw pectins for enzyme production. Polygalacturonase production was high in raw pectin sources like Orange peel (16.8 U/ml) Jack fruit rind (38 U/ml) Carrot peel (36U/ml) and Beet root peel (24U/ml). Selected Aspergillus awamori MTCC 9166 was found to be having good polygalacturonase, xylanase, cellulase and weak amylase and protease activities. This isolate with multi-enzyme production could have application for enzymes production and degradation of fruit and vegetable waste in the process of urban waste disposal.     

Electron microscopic observations of the surface of L-cells in culture

Summary Techniques are described for the preparation of preshadowed replicas of both the upper and lower surfaces of L-cells in culture, and of cross sections of L-cells growing on a cellophane substrate. These revealed long slender microvilli, 800 to 1,100 A in diameter, projecting from both upper and lower surfaces of the cells. These microvilli were frequently observed to contact other cells and substrate, and to leave material behind on the substrate. The plasma membrane of the lower surface was separated from the substrate by an electron-lucent gap 200 to 300 A wide. The surface coat of the L-cell was visualized by staining with colloidal iron and ruthenium. Staining with colloidal iron was most intense on the surface of the microvilli. The gap between cell and substrate was intensely stained with ruthenium red. Enzymatic digestion of living cells revealed that both trypsin and neuraminidase reduced the staining of the cell coat by colloidal iron, whereas only trypsin altered its staining with ruthenium red. After trypsin treatment, fragments of an amorphous material with the staining characteristics of the cell coat were observed between the denuded cells. Treatment with ribonuclease, chymotrypsin or hyaluronidase did not affect the staining of the cell coat.     

Detection of pectic enzymes in pectin-acrylamide gels

A rapid, sensitive slab gel electrophoretic method for the study of pectic enzymes is described. By incorporating pectin in acrylamide gels and subsequent staining with ruthenium red, simultaneous detection of esterases, glycosidases, and lyases was possible due to their differing staining reactions. Application of the method to the study of the thermal stability of enzymes is described. Approximate isoelectric points of the enzymes could be determined from their positions in the gel.     

Ruthenium red as a resonance Raman probe of Ca2+ binding sites in biological materials.

We have observed substantial changes in the resonance Raman spectrum of ruthenium red when it is added to calcium ion binding molecules and organelles, including proteins, phospholipids, chelating agents, and intact mitochondria. The addition of Ca²⁺ ions can reverse these observed spectral changes. In the case of cytochrome c, ruthenium red binding varies with oxidation state in a manner parallel to that for Ca²⁺ binding. The resonance Raman spectrum of a ruthenium red-phospholipid complex shows differences from that of a ruthenium red-protein complex, enabling us to distinguish between binding to these different classes of molecules. Our studies suggest that the primary constituent of the low-affinity Ca²⁺ binding sites in mitochondria is cardiolipin.     

Ruthenium Red-Mediated Osmium Binding for Examining Uncoated Biological Material Under the Scanning Electron Microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% O₃O₄ and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Ruthenium red-mediated osmium binding for examining uncoated biological material under the scanning electron microscope

A simple technique for examining uncoated soft biological material under the scanning electron microscope is described. Rat tissues were initially fixed in 2.5% glutaraldehyde either by intravascular perfusion or by immersion. The samples were placed in buffered 2.5% glutaraldehyde containing 0.05% ruthenium red and postfixed in buffered 1% osmium tetroxide containing 0.05% ruthenium red. The samples were alternately incubated 3 to 5 times in 1% OsO4 and 0.1% ruthenium red solutions with continuous shaking at room temperature. The specimens were dehydrated, critical point dried, mounted and examined under the scanning electron microscope. Contour details were clearly defined at both the external and cut surfaces of the tissues. The specimens could be observed for more than 30 minutes without excessive charging or glow effects and the material remained stable under the beam at 20-25 kV and at various magnifications.     

Ruthenium red as a stain for electron microscopy. Some new aspects of its application and mode of action.

Commercial ruthenium red has been tested for its purity by spectrophotometry. Impurities detected by this method could be abolished by nitric acid-precipitation of ruthenium brown. This substance has no effect on cell surface staining and converts almost completely to ruthenium red under the conditions used in electron microscopy. It was found, by photometric analysis, that in the ruthenium red-osmium tetroxide-cacodylate combination, generally used for cell surface staining, chemical reactions between ruthenium red and osmium tetroxide occur. As aerial oxidation of hexammineruthenium2+ leads to a product with some surface staining capability, it is suggested that an oxidized product of ruthenium red is responsible for binding to cellular components, and that a reduced product of osmium tetroxide gives an additional contrast enhancement. In ruthenium red-osmium dioxide combinations ruthenium red seems to bind to cell surfaces without any molecular alteration, and contrast is gained by the model proposed by Blanquet (1976b). The latter method could open a way for investigating the binding of ruthenium red to certain natural compounds involved in calcium transport, as postulated by a number of authors. Both ruthenium-osmium combinations differ in their cell surface staining ability. The ruthenium red-osmium dioxide combination tends to form distinct subunits, whereas the osmium tetroxide variety stains homogeneously. In combination with osmium dioxide, the surface staining is affected by EDTA, and, in contrast to osmium tetroxide, a successive application of ruthenium red and osmium dioxide as possible.     

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.