Electronmicroscopical detection of iota-carrageenan as its ruthenium red complex

Specific detection of iota-Carrageenan (i-CAR) at the ultrastructural level has been obtained by coupling with ruthenium red (RR) - an electron microscopic stain. The i-CAR-RR complex showed electron density on carbon layers. Peritoneal macrophages were treated with the complex and after 3 h it caused the same morphological changes in macrophages as iota-Carrageenan alone. On the surfaces of macrophages, fine filamentous electron dense material - the i-CAR-RR complex - was detected.     

Coral red fluorescence protein as genetic modified baculovirus tracer

Genetic modified baculovirus (GMBV) are among the most promising alternatives to chemical insecticides. One of the deterrents to the GMBV development is the lack of simple and cost-effective methods for monitoring their efficacy and ecology in fields. Here, we demonstrate the DsRed gene from coral can serve as a convenient GMBV tracer. Insect larvae, including Trichoplusia ni, Spodoptera exigua, and Spodoptera litura, infected the GMBV containing the DsRed gene can emit red fluorescence under sun light without any prosthetic apparatus.     

Positive colloidal thorium dioxide as an electron microscopical contrasting agent for glycosaminoglycans,compared with ruthenium red and positive colloidal iron

Summary In electron microscopy Thorotrast has been used as a specific contrasting agent for acid glycosaminoglycans. Because of its high atomic number, thorium (Z=90) gives good contrast in the electron microscope, but at present it is less frequently used for this purpose. We prepared a positive colloidal solution of ThO₂ without stabilizers to compare its properties with those of ruthenium red and positive colloidal iron for contrasting fetal mouse epiphyseal cartilage. The results indicate that colloidal ThO₂, which is easy to prepare in any laboratory, gives better results than ruthenium red and colloidal iron do in this kind of cartilage. Furthermore, as judged from data in the literature and obtained in our laboratory, it penetrates this tissue better than Thorotrast does, probably because of the absence of stabilizers.     

A Zoletil-Rompun mixture as an alternative to the use of opioids for immobilization of feral red deer

Sixty chemical immobilizations of red deer (Cervus elaphus hippelaphus) have been carried out during an etho-ecological study from August 1994 to December 1996 in a 35 ha pen in the district of Nitra (Slovac Republic). Our objective was to determine the efficacy and standard dosages of Zoletil and Rompun for the immobilization of adult red deer in feral conditions as an alternative to the use of the highly toxic opioids. We therefore compared an Immobilon-Rompun combination (ImRo) with a 1:1 mixture of Zoletil and Rompun (ZoRo) as an injectable solution. Use of both combinations led to the immobilization of >92% of deer with an injection volume <3 ml. Mean (SD) dose to achieve immobilization was 35 (14) microg/kg ethorphine + 0.14 (0.056) mg/kg acepromazine + 0.36 (0.14) mg/kg xylazine compared to 1.2 (0.8) mg/kg tiletamine + 1.2 (0.8) mg/kg zolazepam + 2.3 (1.6) mg/kg xylazine. This corresponds to a volume of 1.8 (0.7) ml/100 kg body mass (BM) for ImRo (range = 1.0 to 4.6) and to 2.3 (1.6) ml/100 kg BM for ZoRo (range = 0.7 to 4.0), respectively. Heart rate, respiratory rate and oxyhaemoglobin saturation values did not differ significantly between the two groups during immobilization. Three deer (5%) died during immobilization, but fatalities could not be directly associated with the drug effect. Mean (SD) time from darting to complete immobilization was 5.5 (4.2) min for ImRo and 7.5 (6.1) min for ZoRo, respectively. Differences were not statistically significant. Anesthesia with both combinations of immobilizing agents could be reversed within 2 min using sarmazenile-yohimbine for ZoRo and diprenorphine-yohimbine for ImXy immobilizations, respectively. We conclude that the 1:1 combination of Zoletil and xylazine is a valuable alternative to the use of opioids for the immobilization of adult red deer including feral adult animals.     

The use of lanthanum chloride as a marker for intercellular junctions in rat exocrine pancreas

A simple technique of perfusion and immersion of tissue in fixative containing lanthanum chloride as an extracellular tracer is described. In addition to functioning as a tracer, the lanthanum chloride appears to enhance electron staining. In rat exocrine pancreas, intercellular spaces between exocrine and centroductular cells were outlined clearly be electron dense material and, at cellular interfaces, spot desmosomes, gap junctions, and tight junctions were demonstrated. The technique proved simple and effective and should prove useful in studies of epithelial and other tissues.     

Selective neurotoxicity of ruthenium red in primary cultures

The inorganic dye ruthenium red (RuR) has been shown to be neurotoxic in vivo when injected intracerebrally. In this work toxicity of RuR was compared in primary cultures of rat cotical neurons, cerebellar granule neurons and cerebellar astroglia. Microscopic examination of the cultures revealed that RuR penetrates the somata of both types of neurosn used and produces vacuolization and loss and fragmentation of neurites. In contrast, no RuR was seen inside cultured astrocytes and no morphological signs of damage were observed in these cells. RuR toxicity wa also assessed by immunocytochemistry of -tubulin and by biochemical measurement of the reduction of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by the cultured cells. The morphological alteratios in the neuros were closely correlated with loss of tubulin immunoreactivity and partivular with a notable decrement in the ability to reduce MTT. Using the latter parameter, it was found that neuronal damage was independent of the age of the cultures, augmented progressively with time of incubation with RuR, from 8 to 24 h, and showed a clear dose-response curve from 20 to 100 M RuR. Astrocytes showed only a slight decrease in MTT reduction after 24 h of incubation with 100 M RuR. It is concluded that RuR seems to be toxic for neurons but not for astroglia, and that this selectivity is probably related to the ability of the neurons to internalize the dye. The possible mechanisms ofRuR penetration and neuronal damage are discussed.Special issue dedicated to Dr. Robert Balázs.     

Adsorption of ruthenium red to phospholipid membranes.

We have measured the distribution of the hexavalent ruthenium red cation (RuR) between water and phospholipid membranes, have shown the critical importance of membrane negative surface charge for RuR binding, and determined the association constant of RuR for different phospholipid bilayers. The studies were performed with liposomes made of mixtures of zwitterionic L-alpha-phosphatidylcholine (PC), and one of the negatively charged phospholipids: L-alpha-phosphatidylserine (PS), L-alpha-phosphatidylinositol (PI), or L-alpha-phosphatidylglycerol (PG). Lipid composition of PC:PX membranes was 1:0, 19:1, 9:1, and 4:1. Liposomes were processed using freeze-and-thaw treatment, and their size distribution was characterized by light scattering and electron microscopy. Experimental distribution isotherms of RuR obtained by ultracentrifugation and spectrophotometry can be reproduced with the Langmuir-Stern-Grahame model, assuming that RuR behaves in the diffuse double layer as an ion with effective valency < 6. In terms of this model, PC-PS, PC-PI, and PC-PG membranes were found to be electrostatically equivalent and the intrinsic association constants of RuR were obtained. RuR has highest affinity to PS-containing membranes; its association constant for PC-PI and PC-PG membranes is about 5 times smaller than that for PC-PS membranes. From the comparison of RuR binding to mixed negatively charged phospholipid membranes and RuR binding to sarcoplasmic reticulum (SR), we conclude that the low-affinity RuR binding sites may indeed be associated with the lipid bilayer of SR.     

Characterization of rhodamine-123 as a tracer dye for use in in vitro drug transport assays

Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.     

Microplate assay for endo-polygalacturonase activity determination based on ruthenium red method

Endo-polygalacturonase (endo-PGase) activity determinations generally rely on viscosity changes or reducing sugar ends produced by this activity over polygalacturonic acid. Torres and coworkers [Enzyme Microb. Technol. 48 (2011) 123–128] showed that ruthenium red (RR) is useful for endo-PGase determination. In this article, we present a high-throughput liquid-based endo-PGase assay based on the RR method and compare it with the viscosity determination method. The reduced assay uses a small volume of enzyme solution, 40 μg of polygalacturonic acid, and 45 μg of RR for each sample determination. Furthermore, we obtained an interconversion factor for RR and viscosity activities.     

A solid-phase immunoassay for human immunoglobulin E: use of 125I-labeled protein A as the tracer

A solid-phase immunoassay has been developed for human immunoglobulin (Ig) E. The specific binding of ¹²⁵I-labeled protein A (¹²⁵I-PA) to the Fc region of rabbit IgG anti-IgE served as a quantitative measure of specific anti-IgE antibody bound to the IgE beads under optimal assay conditions. Inhibition of antibody binding by known amounts of standard IgE was reflected in a decreased binding of ¹²⁵I-PA. The degree of inhibition of ¹²⁵I-PA binding was related to the amount of fluid-phase IgE present and gave a standard curve which was used to determine the concentration of IgE in test samples. The sensitivity of this method and a double antibody radioimmunoassay (RIA), which was developed using the same IgE preparation and anti-IgE antibody, was approximately the same. These assays gave similar results when used to determine levels of IgE in normal human sera that had been absorbed with protein A—Sepharose to remove components responsible for specific and nonspecific interference in the assays.     

Freeze-fracture and tracer studies on the intercellular junctions of insect rectal tissues.

Both rectal pads of the cockroach and rectal papillae of the blowfly possess highly infolded lateral borders; these are associated by desmosomes and septate junctions that maintain the physical integrity of the cell layer at the luminal and basal intercellular regions. Adjacent cells are coupled by gap junctions that allow for cell-to-cell communication and which occur at intervals along the undulating lateral clefts. In rectal pads, occluding basal tight junctions are found as well as extensive scalariform junctions. The latter, like the stacked membrane infoldings of rectal papillae, exhibit intercellular columns and numerous intramembranous P face particles; these are undoubtedly involved in ion transport. In the inter-stack clefts of papillae, reticular septate junctions are encountered which, after freeze-fracture, possess a striking network of PF ridges and EF grooves that are discontinuous and not always complementary. These may serve to regulate the speed and extent of distension of the clefts during solute movement to allow for even and effective fluid flow in this transporting epithelium.