Copper Ferrocyanide Research Articles

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella

Mitochondrial metabolic competence, defined as the organelle's capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions.In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/μm3 of tissue (numeric density: Nv); the mitochondrial volume fraction/μm3 of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

Effective Removal of Cesium and Strontium from Radioactive Wastes Using Chemical Treatment Followed by Ultra Filtration

Chemical treatment assumes an important role in the management of radioactive wastes as it is a simple technique and offers advantage in terms of handling of wastes thereby reducing the risk of mansievert exposure. Low level wastes (LLW) and intermediate level wastes (ILW) are generated in various facets of nuclear fuel cycle and have various chemical composition. A systematic study was carried out by using copper ferrocyanide and calcium phosphate precipitation methods for the removal of cesium and strontium, respectively. The supernatants were subjected to ultra filtration (UF) using a membrane having a pore size of 0.2 μm. The decontamination factors (DF) at 2 and 24-hour intervals with and without UF were estimated. The DF obtained was in the range of 200–300 for cesium and 200 for strontium with LLW solution which has chemical characteristics similar to ground water. Two hours of settling is adequate for strontium before UF. In case of cesium there is no much change in the DF values by UF. However, the UF has helped in the solid — liquid separation as the flocks of copper ferrocyanide precipitate are feathery in nature. The effect of ionic strength and the presence of TBP on the removal efficiency of cesium and strontium have also been studied. DF are observed to be a function of ionic strength and are low in deionized water, in salt solutions containing 1 to 4M sodium nitrate and also in solutions of ILW. However, increasing the chemical dosing to two times of normal plant dosing has yielded a DF of about 200 for sodium nitrate solutions with respect to cesium removal. When the concentration of ammonium nitrate in the waste exceeds 0.1M, the DF reduces. Entrained TBP as well as soluble TBP reduces the removal efficiency of cesium. This paper deals with the experimental data and mechanism of the processes involved in the removal of cesium and strontium.

Glucose-6-phosphate dehydrogenase cytochemistry using a copper ferrocyanide method and its application to rapidly frozen cells.

We describe an improved copper ferrocyanide-based method for cytochemical detection of glucose-6-phosphate dehydrogenase (G6PD), which was used to localize the enzyme within the ultrastructure of rat hepatocytes and adrenocortical cells. With this method, glutaraldehyde fixation and the addition of exogenous electron carriers (for example, phenazine methosulfate) to the cytochemical reaction medium were essential. Copper ferrocyanide reaction product showing the distribution of G6PD was readily recognized at the light microscopic level as Hatchett's brown staining and at the electron microscopic level as electron-dense deposits. Within stained regions, enzyme cytochemical G6PD activity was found to be associated with ribosome-like structures. Because G6PD is a soluble, cytosolic enzyme, its displacement or extraction may occur during conventional fixation. We, therefore, combined a rapid-freezing technique with G6PD enzyme cytochemistry. The resultant rapid-freezing enzyme cytochemistry enabled us to show the subcellular distribution of G6PD in a more life-like state; the localization of G6PD in rapidly frozen cells was in substantial agreement with that in conventionally fixed cells.

Improvement of Cs leaching resistance of solidified radwastes with copper ferrocyanide (CFC)-vermiculite

Cesium removal from de-ionized water, seawater, and limewater using copper ferrocyanide (CFC) and porous media including silica gel, bentonite, vermiculite, and zeolite as adsorbents were investigated; CFC was incorporated with vermiculite to prepare a compound adsorbent for improving the Cs-leaching resistance of solidified borate radwastes. It was shown that the Cs-removal efficiency by CFC, defined as the percentage of cesium removed or adsorpted from solution, was largely affected by pHs of the solutions. Good removal efficiency occurred at pHs ranging from 3 to 12 with the best from 7 to 10. Vermiculite and zeolite were shown to have better removal power than silica gel and bentonite, and vermiculite was chosen to incorporate with CFC to make compound adsorbents because of its good compatibility with CFC floc. Compound adsorbents with different CFC contents were used as additives in the solidification of radioactive borate wastes for improving the cesium leaching resistance of the solidified products. Experimental results showed that the cesium leachability index measured following the method described in ANSI/ANS 16.1 increased from 7.96 to 9.76 by adding 0.25% of a compound adsorbent containing 20% CFC and 80% vermiculite. It indicated that the compound adsorbent is very useful for improving cesium-leaching resistance of the solidified borate wastes.

Morphometry and acetylcholinesterase activity of the myenteric neurons of the mouse colon in the chronic phase of experimental Trypanosoma cruzi infection.

The myenteric plexus of the proximal colon, midcolon, and distal colon was studied in mice chronically infected with the Y strain of Trypanosoma cruzi by means of histochemical methods for NADH-diaphorase and acetylcholinesterase (AChE) on whole mount preparations. Ganglia of infected mice displayed an irregular distribution, with neurons severely altered in form and were found side by side with slightly degenerated or morphologically normal ones. Significant reductions of at least 36% in the numbers of neurons were recorded in all regions of the colons of infected animals, especially in the distal colon where the neuron number decreased by more than 44%. Measurements of neuron size suggest that the neuronal destruction caused by T. cruzi affected the medium and large neurons. The small neurons apparently were not affected by the infection. The histochemical demonstration of AChE by the direct coloring copper ferrocyanide method showed that in the control animals, most of the neurons of the plexus displayed AChE activity in the cytoplasm although the neurons showed different reaction intensities. The AChE activity was also present, but at a lower intensity, in the myenteric plexus of the colons of infected animals. These results suggest that the T. cruzi infection affects some categories of neurons and implies that some particular enteric neurotransmitter systems could be affected and the potency of their action upon intestinal function consequently reduced.

Copper ferrocyanide — polyurethane foam as a composite ion exchanger for removal of radioactive cesium

A method has been developed for the removal of cesium from the aqueous radioactive waste using a composite ion-exchanger consisting of Copper-Ferrocyanide Powder (CFC) and Polyurethane (PU) Foam. Polyvinyl acetate has been used as a binder in the preparation of CFC-PU foam. The physical properties of CFC such as density, surface area, IR stretching frequency and lattice parameters have been evaluated and also its potassium and copper(II) content have been estimated. Optimization of loading of CFC on PU foam has been studied. The CFC-PU was viewed under microscope to find out the homogeneity of distribution. Exchange capacities of the CFC-PU foam in different media have been determined and column studies have been carried out. Studies have been undertaken on extraction of cesium from CFC foam and also on digestion of spent CFC-PU foam and immobilization of digested solution in cement matrix. The cement matrices have been characterized with respect to density, bio-resistance and leaching resistance.

Augmented placental production of leptin in severe preeclampsia

The present study investigated the localization of glucose-6-phosphate dehydrogenase (G6PD) activity in the human placenta at various gestational ages. Placentae from patients with severe pre-eclampsia were also studied. Ultrastructural enzyme–histochemical analysis was performed by newly developed G6PD histochemistry using copper ferrocyanide as a capturing agent. Precipitates indicative of G6PD activity were markedly visible in the cytotrophoblastic cytoplasm and faintly in the syncytiotrophoblastic cytoplasm of placentae at various gestational ages, as well as those from pre-eclampsia. Frequently, precipitations were localized on the cytosolic side of the endoplasmic reticular membranes of the cytotrophoblasts. Stringent cytochemical control experiments performed also ensured the specific detection of G6PD activity. The results indicated that cytochemically detectable G6PD was localized in cytotrophoblastic cytoplasm. This enzyme may play significant roles in the carbohydrate metabolism of the human placenta, and further maintenance of villous tree architecture. Combining the previous data, the human placenta has many carbohydrate metabolizing-enzymes, similar to the adult liver.

Ultracytochemistry of 3beta-hydroxysteroid dehydrogenase in Leydig cell precursors and vascular endothelial cells of the postnatal rat testis.

In the biosynthesis of steroid hormones 3beta-hydroxysteroid dehydrogenase (3beta-HSD) is a key enzyme. The present report describes the subcellular localization of the enzyme in the fetal-type Leydig cells, the fibroblast-like precursors of adult-type Leydig cells and in endothelial cells of interstitial capillaries. Histochemical methods for light microscopy and ultracytochemical methods for electron microscopy were used on rat testes of postnatal day 15. 3beta-HSD reactivity was located at subcellular levels by means of the ferricyanide method. A specific, distinct localization of reaction product in the form of copper ferrocyanide precipitates was observed on the membranes of the smooth endoplasmic reticulum not only in the fetal-type Leydig cells and the fibroblast-like precursors of adult-type Leydig cells, but also focally in the endothelial cells of interstitial blood capillaries. Topographically, the 3beta-HSD-positive precursors were most often found in the outer layer of the boundary tissue and surrounding interstitial blood vessels. The capillaries with 3beta-HSD-positive endothelial cells were usually located in the vicinity of 3beta-HSD-positive Leydig cells. For the first time, 3beta-HSD has been located at the subcellular level in precursors of adult-type Leydig cells and focally in capillary endothelial cells associated with them. Due to the close association between 3beta-HSD-positive vascular endothelial cells and Leydig cells a paracrine relationship between the two cell types may be involved in the acute regulation of steroidogenesis by blood-borne luteinizing hormone.

Selective Removal of Cesium from Acid Solutions with Immobilized Copper Ferrocyanide

Copper ferrocyanide can be immobilized on Chelex-20, a technical grade chelating resin, for selective removal of cesium from neutral to acidic solutions. The immobilized copper ferrocyanide is much easier to handle than the copper ferrocyanide powder in extraction and separation of cesium from aqueous solutions. In the presence of a reducing agent such as hydrazine, the immobilized copper ferrocyanide resin is able to remove 98% of the cesium from a simulated acid waste solution in one cycle of batch experiments. The resin shows promising properties for remediation of acidic nuclear wastes and for concentration of (137)Cs from environmental samples for quantitative analysis.

Impaired succinic dehydrogenase activity of rat Purkinje cell mitochondria during aging

The perikaryal Purkinje cell mitochondria positive to the copper ferrocyanide histochemical reaction for succinic dehydrogenase (SDH) have been investigated by means of semiautomatic morphometric methods in rats of 3, 12 and 24 months of age. The number of organelles/ μm 3 of Purkinje cell cytoplasm (Numeric density: Nv), the average mitochondrial volume (V) and the mitochondrial volume fraction (Volume density: Vv) were the ultrastructural parameters taken into account. Nv was significantly higher at 12 than at 3 and 24 months of age. V was significantly decreased at 12 and 24 months of age, but no difference was envisaged between adult and old rats. Vv was significantly decreased in old animals vs. the other age groups. In young and old rats, the percentage of organelles larger than 0.32 μm 3 was 13.5 and 11%, respectively, while these enlarged mitochondria accounted for less than 1% in the adult group. Since SDH activity is of critical importance when energy demand is high, the marked decrease of Vv supports an impaired capacity of the old Purkinje cells to match actual energy supply at sustained transmission of the nervous impulse. However, the high percentage of enlarged organelles found in old rats may witness a morphofunctional compensatory response.

96/05531 Fixation of copper ferrocyanide cake in the urea-formaldehyde matrix

96/05531 Fixation of copper ferrocyanide cake in the urea-formaldehyde matrix

Determination of 15N/14N and 13C/12C in Solid and Aqueous Cyanides

The stable isotopic compositions of nitrogen and carbon in cyanide compounds can be determined by combusting aliquots in sealed tubes to form N2 gas and CO2 gas and analyzing the gases by mass spectrometry. Free cyanide (CN-aq + HCNaq) in simple solutions can also be analyzed by first precipitating the cyanide as copper(II) ferrocyanide and then combusting the precipitate. Reproducibility is ±0.5‰ or better for both δ15N and δ13C. If empirical corrections are made on the basis of carbon yields, the reproducibility of δ13C can be improved to ±0.2‰. The analytical methods described herein are sufficiently accurate and precise to apply stable isotope techniques to problems of cyanide degradation in natural waters and industrial process solutions.

Quantitative study and architecture of nerves and ganglia of the rat heart.

The cardiac plexus of the Wistar rat was investigated in whole-mount preparations of the atria by NADH-diaphorase staining and by the acetylcholinesterase (AChE) histochemical technique. The plexus lies over the muscular layer of the atria, dorsal to the muscle itself, in the connective tissue of the subepicardium. The plexus contains on average 975 +/- 150 neurons, occurring singly or gathered in packed ganglia. The ganglia are found regularly in certain situations, namely, cranially to the pulmonary veins (44% of total); cranially and to the left of superior vena cava (10%); in the interatrial groove (21%); to the left of the left pulmonary vein (11%); caudally to the pulmonary veins (12%) and in the wall of the coronary sinus (1%). Ganglia are never found on the auricular appendages. For the histochemical demonstration of AChE, the 'direct coloring' copper ferrocyanide method was used. Extrinsic nerves enter the atria along the superior vena cava, along the pulmonary veins and along the coronary sinus, forming ganglion-containing plexuses. From specific sites of these plexuses, nerves proceed to the ganglia located to the left of the superior vena cava, to the ventral and dorsal ventricular wall and to the wall of the right atrium, where they form a delicate plexus accompanying the muscle fibers. Most of the neurons of the plexuses displayed AChE activity in the cytoplasm though they presented different reaction intensities. The distribution of cardiac nerves from groups of neurons located at discrete sites may indicate that postganglionic nerves selectively project to and thus control specific cardiac regions and/or functions.

Fixation of Copper Ferrocyanide Cake in the urea-formaldehyde matrix

Fixation of Copper Ferrocyanide Cake (CFC), which is generated from the chemical treatment plant of low-level radioactive liquid effluent in a urea-formaldehyde (UF) matrix, was carried out. Boric acid was used as a catalyst for the UF polymerization. The physical properties such as compressive strength and diffusion coefficient were evaluated for UF waste forms and were compared with those of Ordinary Portland Cement (OPC) waste forms. While the compressive strengths of UF and OPC waste forms were comparable, the former showed diffusion coefficients about two to three orders lower. Almost all the cesium was released from the OPC waste form that contained less than 18% of CFC by weight in the final waste form. The release of cesium from the OPC waste forms was high due to the presence of high alkalinity and calcium ions in the cement. To find out the temperature rise in the preparation of UF waste form, blocks having a size of about 5 1 were prepared; it was observed that there was no increase in peak center temperatures during the curing process.

Role of metal ferrocyanides in chemical evolution

Adsorption of several ribose and 2′-deoxyribose 5′-nucleotides on zinc- and copper ferrocyanides has been studied at a neutral pH of 7.01. The Langmuir adsorption isotherm was used to determine the values of KL and Xm. Both types of nucleotides, ribose and 2′-deoxyribose, showed similar adsorption behavior on zinc- and copper ferrocyanides. Zinc ferrocyanide showed larger adsorption as compared to copper ferrocyanide. Purine nucleotides adsorbed more than pyrimidine nucleotides on both the metal ferrocyanides probably because of an additional binding site in the imidazole ring in purines. Results of the present study suggest the importance of metal ferrocyanides and metal ions in stabilization of nucleotides during processes of prebiotic condensation reactions.

Flocculation studies on freshly precipitated copper ferrocyanide for the removal of caesium from radioactive liquid waste

Flocculation of Copper ferrocyanide precipitate, used for the removal of Cs-isotopes from low-level and intermediate-level radioactive liquid waste, has been studied. Application of optimum dosages of flocculants, such as Polyelectrolytes and Fe 3+ ions, is observed to enhance the removal of Cs and aid the separation of solid and liquid phases. Electrophoretic measurements have been used as a tool to determine the optimum dose of ferric ions by finding out the “reversal of charge concentration (RCC)” for Cu-ferrocyanide, precipitated in effluents of different specific conductances. The optimum requirement of Fe 3+ ions increases with increasing specific conductances of the effluents. Presence of a complexing agent like EDTA affects the removal of Cs and also the separation of phases. The problem can be solved, at least for low concentration of EDTA, by lowering the pH to an optimum value, which has again been determined through electrophoretic measurements. It is inferred that uptake of Fe 3+ ions by Cu-ferrocyanide proceeds through “adsorption” and “ion-exchange” with Cu 2+ ions. When Cs + is present at very low concentration, for example as a radiopollutant, its removal is favoured on addition of Fe 3+ as flocculant, but at higher concentrations, the Cs + ions also partially undergo “exchange” with the Cu 2+ ions, thus participating in the formation of precipitate. Addition of Fe 3+, then, may not be desirable, as it may exchange with both Cu 2+ and Cs + ions, releasing them into solution.

Coordination properties of polymeric azacrown ethers

New polymeric aza-crown ethers (PACE), soluble in water and organic solutions and cross-linked ones, containing different macrocycles in side chains have been synthesised by polymerisation and chemical modification reactions. The coordination properties of these PACE are studied. It is shown that during coordination of the VO2+, Cu2+ ions with PACE, cross-linking of macromolecules by metal ions takes place by “sandwich” complex formation. The character of complexes distribution along PACE coils may be equal or nonequal, depending on polymer reactivity. The synthesised PACE are used as high specific polymer reagents for colloid rare metals recovery. It is shown that flocculation of negatively charged inorganic sols with PACE is due to electrostatic adsorption and specific binding. In acid and neutral media, dispersions of gold, silver, copper ferrocyanide and silicon dioxide undergo high flocculation as a result of non-specific electrostatic adsorption. Alkaline media provide conditions for donor-acceptor binding, increasing flocculation selectivity. In the uncharged state (pH 11,2) PACE exhibit high selectivity in binding gold particles. Stability of colloidal dispersions in the presence of monomer aza-crown ether (ACE) indicates an important polymer influence on the fine metal particles flocculation.

A study on the Ogawa's system for cytochemical demonstration of oxidoreductases with ferricyanide as electron acceptor.

Spectrophotometric study on kinetics of the reaction under the conditions mimicking its course in the tissue, carried out with α-glycerophosphate as substrate, rat spermatogenic mitochondria as a source of enzyme, tris/hydroxymethyl/aminomethane as copper complexing agent, and in pH 8.4, i. e. in such a range in which the final product, copper ferrocyanide, is soluble led to the following conclusions. Enzymatic oxidation of the substrate is immediately followed by ferricyanide reduction, whereas the final product preciptation starts with a few minutes latency. Dimethylsulfoxide (DMSO) in the final concentration of 5% does not alter the reaction course. However, at its concentration of 15% there is no latency in the final product formation that runs parallelly to the course of the reduction of ferricyanide but there is an inhibition of both the enzymatic reduction of ferricyanide and the formation of copper ferrocyanide as final reaction product. On fine structural level it was found that already at 5 per cent concentration of DMSO and at a shortened time of incubation the localization of the reaction for a-glycerophosphate: ferricyanide oxidoreductase in mitochondria and the other structures of rat spermatids was improved.

Ultracytochemistry of glucose-6-phosphate dehydrogenase in adrenal gland.

The distribution of glucose-6-phosphate dehydrogenase (G6PD) activity has been studied by a copper ferrocyanide method in the adrenal cortex cells of a rat. The site of the G6PDH activity was close to the ribosome between the round mitochondria of zonas fasciculata and reticularis.

Double staining procedure for histochemical localization of alcohol and aldehyde dehydrogenase activities in the mouse liver.

The double staining procedure using both the nitroblue tetrazolium (NBT) and copper ferrocyanide methods was presented for the simultaneous localization of two different dehydrogenase activities in a single histologic section.Sections of normal mouse liver were stained for alcohol dehydrogenase (ADH) activity using the NBT method first, then stained for aldehyde dehydrogenase (ALDH) activity using the copper ferrocyanide method. However, the formazan showing ADH activity must chelate metal ion (Cu2+, Co2+, or Ni2+) prior to staining for ALDH activity.Both the blue metal-formazan complex showing ADH and brown copper ferrocyanide showing ALDH activities in a single liver section were localized mainly in the perivenous zone of the hepatic lobule. In addition, ALDH activity is always localized in the zone of the lobule where ADH activity is present.