Rubeanic Acid Research Articles

Contraction of neuronal branching volume: an anatomic correlate of Pavlovian conditioning.

Associative memory of the mollusc Hermissenda crassicornis, previously correlated with changes of specific K+ currents, protein phosphorylation, and increased synthesis of mRNA and specific proteins, is here shown to be accompanied by macroscopic alteration in the structure of a single identified neuron, the medial type B photoreceptor cell. Four to five days after training, terminal arborizations of B cells iontophoretically injected with Ni2+ ions and then treated with rubeanic acid were measured with charge-coupled device (CCD)-digitized pseudocolor images of optical sections under "blind" conditions. Boundary volumes enclosing medial-type B-cell arborizations from classically conditioned animals were unequivocally reduced compared with volumes for naive animals or those trained with unpaired stimuli. Branch volume magnitude was correlated with input resistance of the medial type B-cell soma. Such associative learning-induced structural changes may share function with "synapse elimination" described in developmental contexts.

Comparison of three histochemical staining methods for the detection of copper in white perch ( Morone americana) with abnormal hepatic copper storage

A comparison of orcein, rubeanic acid and rhodanine histochemical stains in serial sections of liver from white perch (Morone americana) with abnormal hepatic copper storage is described. Staining reactions correlated well with hepatic copper concentrations although the threshold was the most problematic for rhodanine and rubeanic acid. Orcein was positive at a lower concentration than the two other stains. Hepatocellular granules and macrophage centres increased in size and number with copper concentration and could be related to increased lysosomes, as described for other species.

Histochemical localization of zinc and copper in rat ocular tissues

Zinc and copper were histochemically localized in rat ocular tissues. Zinc was demonstrated by the dithizone method, and copper by the rubeanic acid and rhodanine methods. The retinal photoreceptor's outer segment showed the presence of zinc, but no zinc reaction was seen in other parts of the retina or other ocular tissues, even in rats fed excess zinc. It seems that photoreceptor cells contain the highest concentration of zinc. Copper was seen in the corneal epithelium and endothelium, iris, ciliary body, lens epithelium, retinal outer nuclear layer, photoreceptor inner and outer segments, retinal pigment epithelium, choroid, sclera, and optic nerve. These trace elements seems to play roles as components of some metalloenzymes and may have other functions till now unknown.

Celiptium-induced nephrotoxicity and lipid peroxidation in rat renal cortex

The antitumor drug celiptium, or N2-methyl-9-hydroxyellipticinium (NMHE), is an ellipticine derivative used in the treatment of breast cancer. Celiptium-induced dose-dependent renal toxicity in rats is characterized by tubular necrosis, tubulo-interstitial lesions and lipid overload in proximal tubular cells. Since biooxidative activation of celiptium occurs in kidney via highly electrophilic intermediates, we studied the effects of celiptium on rat renal cortex lipids in the context of lipid peroxidation damage. Female Wistar rats were injected with a single i.v. dose of 20 mg/kg celiptium and were killed on day 2, 4 or 8. Histochemical analysis of kidney sections detected Oil Red O (ORO)-positive deposits, whereas the same sections studied using Holczinger's copper rubeanic acid method showed free fatty acid (FFA) granules in renal tubular cells of celiptium-treated rats. Electron microscopy revealed large fatty droplets in proximal tubular cells. As creatinine clearance decreased on days 4 and 8, celiptium induced a significant increase in renal cortex FFA levels (6-fold increase over pretreatment values on day 8), whereas total glycerides increased 1.5 times. A 15% decrease in total phospholipids (PL) and a 50% decline in the mass of phosphatidylethanolamine (PE) were detected by lipid phosphorus assay. A 1.2-fold decrease in the unsaturation index of total PL was noted, with a significant decline in arachidonic acid (20:4). A 15% decrease in arachidonic content was observed in the fatty acid composition of PE. Analysis of the fatty acid composition of neutral lipids showed changes only in the FFA class. A great proportion of oleic (18:1) and linoleic (18:2) acids was found. Iodometric titration and thiobarbituric acid (TBA) reactivity detected respectively significant amounts of lipid hydroperoxides and TBA-reactive material in renal cortex lipid extracts on days 2, 4 and 8. The lipid-peroxidation process appeared to be involved in the pathogenesis of celiptium nephrotoxicity.

Metallothionein and copper in liver disease with copper retention—a histopathological study

We have examined the relationship between (a) histochemically demonstrable copper using rubeanic acid, (b) copper-associated protein (CAP) using orcein, and (c) immunoreactive metallothionein (MT) using DNP hapten sandwich staining and have correlated these with histological lesions in 95 liver biopsies from patients with conditions associated with hepatic copper retention, 4 fetal livers, and 25 histologically normal adult controls. No copper or CAP was present in normal adult liver but periportal CAP was present in fetal liver. MT was present in hepatocytes of normal livers with a predominantly perivenular (centrilobular) cytoplasmic distribution varying in staining intensity; all fetal hepatocytes stained strongly for MT. Fifty-two of 95 (55 per cent) abnormal livers contained CAP and 42 (44 per cent) contained both CAP and copper. In CAP-positive livers, the commonest histological lesions were piecemeal necrosis and cholestasis. CAP was present in (a) 15/15 cases of primary biliary cirrhosis including early cases with minimal pathology; and (b) 5/5 cases of Wilson's disease, 6/6 cases of biliary atresia, and 3/9 cases of sclerosing cholangitis. In other conditions, it was present in 25-50 per cent of cases. MT distribution was abnormal in most CAP-positive livers. Necrotic hepatocytes were intensely MT-positive and in Wilson's disease had a characteristic appearance.

Liver cell carcinoma with orcein‐positive granules and no identifiable copper

A liver cell carcinoma removed surgically by partial hepatectomy from a 79-year-old woman contained abundant orcein-positive granules in many tumour cells. The granules were similar to those usually identified as representing copper-associated protein, yet they lacked copper, as demonstrated by negative rhodanine and rubeanic acid stains and energy dispersive X-ray microanalysis.

Electrophysiology of K+ Transport by Midgut Epithelium of Lepidopteran Insect Larvae: II. the Transapical Electrochemical Gradients

ABSTRACT The apical surface of the midgut of Manduca sexta larvae is composed of the apical membranes of columnar cells, in the form of microvilli, and the apical goblet of goblet cells. Considerable evidence has suggested that the apical electrogenic pump that is responsible for transepithelial K+ transport is located on the apical membrane of goblet cells. In the present study the transapical potentials and K+ chemical activity [(K+)] gradients of columnar and goblet cells of posterior midgut were examined in the short-circuited gut. In some experiments the recording site was localized by ionophoresis of NiCl2 followed immediately by fixation in rubeanic acid. The (K+) of goblet cavities was substantially higher than that of the free solution on the gut luminal side (mean value of 94mmol 1-1 in standard bathing solution). The goblet cavity was electrically positive to the gut lumen (mean value of 40 mV in standard bathing solution). When the rate of pumping of K+ into the goblet cavity was decreased by hypoxia or decreased bathing solution [K+], the electrical potential gradient between cytoplasm and goblet cavity decreased while intracellular (K+) and goblet cavity (K+) were relatively stable. These studies provide evidence that a negatively charged goblet matrix is present in goblet cavities. Furthermore, they suggest that it is the voltage-sensitivity of the apical pump to the electrical component of the transapical electrochemical gradient, and not a concentration-dependence of the pump, that exercises the major role in determining the relationship between extracellular (K+) and net K+ transport by the isolated gut.

Histochemistry as a tool for examining possible pathologic cause-and-effect relationships between heavy metal and inflammatory lesions in oysters, Crassostrea virginica

Unexplained inflammatory lesions have been seen on many occasions in oysters ( Crassostrea virginica) that have been examined by histopathologic methods. Application of the Mallory's hematin and the rubeanic acid methods for demonstration of copper in histologic sections revealed strong staining reactions in granules of some, but not all, granular hemocytes. These cells were clearly involved as constituents of acute inflammatory lesions characterized by infiltration of arteries, occlusion of veins, and diapedesis through external epithelia of the mantle and gastrointestinal tract. Heavily contaminated sites (i.e. Raritan Bay, New Jersey) showed strong staining reactions in a large number of animals examined. Control sites such as Great Bay, New Jersey, showed much lighter reactions. Seasonal sampling in the New Jersey sites revealed the most intense involvement in the late summer with apparent decreasing staining intensities in the fall. Other species of bivalve mollusks ( Mytilus edulis and Mercenaria mercenaria) only showed significant staining reactions for heavy metal in kidney tissues with no evidence that heavily contaminated sites had higher levels. These results constitute preliminary evidence of a cause-and-effect relationship between a heavy metal contaminant (most probably copper) and inflammatory lesions in the oyster.

Evaluation of histochemical methods for the detection of copper overload in rat liver

Histochemical methods have invariably shown a good correlation with copper analysis by absorption spectrophotometry in the identification of canine copper storage diseases. But, in Wilson's disease (WD) in humans no such correlation exists and similar discrepancies have also been observed in copper-loaded rats. This study attempts to quantify stainable copper in the livers of copper-loaded rats and relate this to the hepatic copper concentrations. Male rats fed a high copper diet (1500 ppm) for 16 weeks were killed at intervals. The livers were analysed for copper and graded according to stainable copper present in paraffin sections stained with rhodanine and rubeanic acid. Initially there was a good correlation between histochemically demonstrable copper and its total concentration, but subsequently, when high liver levels of the metal were present, copper staining was very variable. This unrealiability has similarities with WD, in which the higher hepatic concentrations of presymptomatic patients are difficult to detect by conventional copper stains. The variation in the binding of copper and its intracellular localisation suggested by these results may have considerable significance in the pathogenesis of copper storage diseases.

Histochemical and ultrastructural study of copper-binding protein in hepatocellular carcinoma

Orcein-positive hepatocellular material (OPHM), found in 18 of 44 hepatocellular carcinomas (HCC), was compared histochemically and ultrastructurally with similar granular materials in other liver diseases. OPHM was not found in 15 control livers, 45 metastatic liver cancers or ten cholangiocellular carcinomas. OPHM in HCC was stained with orcein when the tissue sections were preoxidized. In addition, it was positively stained with rhodanine and rubeanic acid for copper. These findings were identical to the OPHM in Wilson's disease, in primary biliary cirrhosis and in neonatal livers. Seven of 12 resected HCC revealed cytoplasmic dense bodies ultrastructurally in which an x-ray microanalyzer demonstrated copper. OPHM in HCC, a granular accumulation, was concluded to be rich in copper-protein complex, and might correspond to dense bodies derived from lysosome ultrastructurally. The pathogenesis and biologic significance of this copper overload in a number of HCC are also discussed.

Uneven copper distribution in the human newborn liver

The pattern of copper distribution in human newborn liver was investigated by histochemical methods (rhodamine, orcein and rubeanic acid) and by atomic absorption spectroscopy. A significant correlation (p less than 0.005) was found between the degree of histochemical positivity and the copper concentration found by atomic absorption spectroscopy. In the majority of the 30 livers examined (first group), the copper concentration was much higher than that of normal adult liver, although exhibiting striking individual differences. No correlation between the copper content and sex, body weight or gestational age was found. From a second group of five livers, longitudinal tissue slices 0.5 cm thick were partitioned into regular blocks of about 0.5 gm, which were individually analyzed by atomic absorption spectroscopy. Copper appeared unevenly distributed within each liver, with marked differences even between adjacent blocks. However, a consistent tendency of copper to accumulate in the left lobe more than in the right one was evident. Five additional blocks, one for each liver, were further partitioned into 10 small specimens of a final size (0.05 gm), comparable to that of a needle biopsy. Even at this sampling level, consisting of tissue fragments taken from a small tissue area, the copper concentration appeared quite irregularly distributed. These findings may be considered for two different aspects: (a) the biological implications of the pattern of copper accumulation in different lobar and lobular liver compartments and (b) the statistical inference, for diagnostic purposes, of the mean liver copper content from measurements of single percutaneous biopsy specimens.

Backscattered electron imaging as a tool for histochemically localizing calcium oxalate with the scanning electron microscope

The scanning electron microscope and a backscattered electron detector were used for the first time to identify calcium oxalate in leaf tissues after labelling crystals with silver. Freehand sections fixed in formalin – acetic acid – alcohol or glutaraldehyde were treated with 5% acetic acid to remove background calcium carbonate and calcium phosphate and were silver stained by calcium-substitution techniques. Two standard staining techniques, Pizzolato (AgNO3–H2O2) and Yasue (AgNO3 – rubeanic acid) tests, were compared. Druses and raphides were identified morphologically and histochemically in the leaves of Anthurium scherzeranum and Dieffenbachia maculata. The Pizzolato procedure was the best method for staining calcium oxalate for scanning electron microscope studies. Excessive deposition of granular material in the Yasue staining method tended to obscure crystal morphology in scanning electron microscope preparations.

Neuronal structure and synaptic distribution of a uropod closer motor neuron in the crayfish terminal ganglion.

One of the uropod closer muscles in the crayfish, the adductor exopodite, is innervated by two large identified motor neurons. They were injected intracellularly with horseradish peroxidase or nickel chloride to reveal the structure and distribution of the input and output synapses using electron microscopy. The development of nickel with rubeanic acid greatly improved the tissue preservation at the ultrastructural level compared with ammonium sulphide. Cell bodies of the motor neurons lying in the ventro-lateral cortex of the ganglion are extensively invaginated by glial cells. Input synapses occur directly upon the primary neurite within the neuropil or upon the major anterior neurite. They are most abundant, however, upon the numerous smaller neurites of the motor neuron. The primary neurite in the dorsal region of the neuropil, upon which no synapses were made, is wrapped with glial cells. Occasionally, these two adductor exopodite motor neurons were found as adjacent postsynaptic profiles at the same synapse when both cells were stained simultaneously in the same preparation. In the present study we could not locate any sites of synaptic output which strictly fulfil the structural criteria of a synapse on the processes of the motor neuron. This result is inconsistent with physiological evidence which suggests that spikeless interactions occur between the two adductor exopodite motor neurons and their synergists. This might be the result of two possible features of the interaction: the sites of synaptic output may be limited to a few restricted branches, and the interaction between these motor neurons may depend largely upon electrical synapses.

Histochemical study of the accumulation of copper in the liver of sheep

Six Merino sheep were dosed orally with a 0.2 per cent solution of copper sulphate, six others were undosed controls. Liver biopsies were obtained and stained for copper by the p-dimethylaminobenzylidene rhodanine (DMABR), rubeanic acid (RA) and ferricyanide (FCN) methods for examination by light microscopy. The initial and most marked accumulations of copper were found within the hepatocytes of the centrilobular zones. Increased copper loading resulted in copper deposition extending through the midlobular to the periportal zones. The deposition of copper was unequal between hepatocytes and with increasing copper loading isolated hepatocytes became packed with copper containing granules. Copper appeared within Kupffer cells and macrophages of portal triads. The first Kupffer cells to be positively stained and the greatest number of such cells were adjacent to the central veins. Accumulation of copper was demonstrated with hepatocytes at copper concentrations equivalent to 44.3 micrograms copper g-1 liver wet weight. The FCN method provided a more satisfactory demonstration of intracellular copper than the RA technique and the latter was better than the DMABR method. However, the DMABR technique provided the clearest morphological details.

Synthesis of Copper(II)–Dithiooxamide Complex in Zirconium Phosphate

Abstract Copper(II)–dithiooxamide (rubeanic acid) complex was synthesized in the interlayer region of zirconium phosphate. The product, called ZP–CuLra, was chracterized by chemical analysis, thermal analysis, infrared spectroscopy, and X-ray diffractometry. The chemical composition of ZP–CuLra was represented as ZrCu(H2NCS)2(PO4)2·H2O or ZrCu(HNCS)2(HPO4)2·H2O. The interlayer distance of ZP–CuLra was found to be 9.0 Å. By analogy with Cu(II)–rubeanic acid complex prepared in a solution, a structural model of ZP–CuLra was proposed.

Separation and detection of traces of copper, iron and manganese in cotton materials by TLC

A TLC method for the fast detection of copper, iron and managanese ions in cotton materials is described. The optimal solvent system is 8∶1∶2 (v/v), acetone: HCl:H2O and the locating reagent is rubeanic acid followed by exposure to ammonia vapour. It was found that in cotton materials, metal ions can be detected at a lower limit of 20 μg per gram of material.

Copper and hepatocellular carcinoma.

The presence of copper and copper-binding protein (CBP) within tumor cells was searched by histochemical methods (rhodanine, rubeanic acid and Shikata's orcein) in a group of 39 autopsies, all consecutive cases of hepatocellular carcinoma (HCC) associated with cirrhosis (HCC + C). In 2 cases, fibrolamellar carcinoma (FLC) not associated with cirrhosis was observed at surgery. Considerable amounts of copper and CBP were found within tumor cells only in the 2 FLC cases and in 1 HCC + C case, in a portion of the tumor showing FLC-like features. Copper-positive tumor cells had an oncocytic appearance, which was confirmed by ultrastructural examination. In 64% of the HCC + C cases (25 out of 39), copper and CBP deposits were found in nonneoplastic hepatocytes mainly distributed along the periphery of cirrhotic nodules. The present study indicates that the storage of copper inside tumor cells is a peculiarity of the FLC type of HCC with a close relationship to the oncocytic nature of neoplastic hepatocytes. The significance of copper deposits in nonneoplastic cirrhotic hepatocytes remains a matter for further investigations.

X-ray fluorescence spectrometric determination of copper in pure iron after precipitation.

Trace amount of copper (II) was quantitatively precipitated with rubeanic acid from aqueous solution of pH 6.06.4. A linear calibration curve for the X-ray fluorescence determination was obtained over the range 0.150 μg of copper. The relative standard deviation of the determination was 1.4% in five runs for 10 μg of copper, and the sensitivity was 60 cps/μg Cu. The following amounts of foreign ions were tolerated in the determination of 20μg of copper : Fe(III) 500 mg, Co(II) 5 mg and Ni(II) 1 mg. The procedure is as follows : Dissolve a sample in hydrochloric acid and hydrogen peroxide. Add 5 ml of 40% tartaric acid solution, adjust the pH to 6.06.4 with aqueous ammonia, and then add 2 ml of 0.1 % rubeanic acid solution. Collect the resulting precipitate on a membrane filter (25 mm, 0.2 μm) and wash with water. After drying the precipitate at room temperature, measure the X-ray intensity of Cu Kα line (2θ=45.05°) and that of background at an angle (2θ) 46.0°, in a vacuum for 40 s by using a molybdenumtarget tube (50 kV, 30 mA), a LiF (2 0 0) crystal and a scintillation counter.

Study in aqueous media of the system technetium — Rubeanic acid analytical applications

The interaction between technetium and rubeanic acid is described and a simple and highly selective spectrophotomemtric determination of techneitum is described. The absorption maximum of the species occures at 675 nm., The molar absorptivity being 5.0·103 1·mol−1·cm−1. The reaction has been investigated and the experimental variables and interferences have been studied.

ChemInform Abstract: METAL COMPLEXES OF RUBEANIC ACID. 3. LARGE‐ANGLE X‐RAY SCATTERING STUDIES OF AMORPHOUS COPPER(II) AND NICKEL(II) COMPLEXES

Chemischer InformationsdienstVolume 16, Issue 42 Physical Organic Chemistry ChemInform Abstract: METAL COMPLEXES OF RUBEANIC ACID. 3. LARGE-ANGLE X-RAY SCATTERING STUDIES OF AMORPHOUS COPPER(II) AND NICKEL(II) COMPLEXES M. ABBOUDI, M. ABBOUDISearch for more papers by this authorA. MOSSET, A. MOSSETSearch for more papers by this authorJ. GALY, J. GALYSearch for more papers by this author M. ABBOUDI, M. ABBOUDISearch for more papers by this authorA. MOSSET, A. MOSSETSearch for more papers by this authorJ. GALY, J. GALYSearch for more papers by this author First published: October 22, 1985 https://doi.org/10.1002/chin.198542052AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume16, Issue42October 22, 1985 RelatedInformation