A comparison of various procedures for fine grain development in electron microscopic radioautography.

Fine grain development for electron microscopic radioautography was investigated with two types of radioactive specimens: sections of tritiated methacrylate, which provide a homogeneously labeled source for quantitative evaluation of the radioautographic reaction, and sections of 125I-labeled thyroid. Radioautographs were prepared with Ilford L4, Sakura NR-H2, Agfa-Gevaert NUC 307 or Kodak NTE emulsions. The radioautographs were developed with one of several "solution physical" development procedures (Agfa-Gevaert, phenidone-ascorbic acid, p-phenylenediamine developers) or with arrested "direct" developments (D-19b, Elon-ascorbic acid developers). By arresting each development at an early stage of the reaction and at progressively longer time intervals, it was possible to examine the sequence of shapes in the growth of developed silver deposits for each emulsion-development combination. Thus, conditions which resulted in the development of small, round, compact silver deposits were defined for each emulsion. These developments were used in conjuction with gold latensification, a treatment which increases the sensitivity of the emulsions and thus compensates for the lowered sensitivity of fine grain development procedures. The location of the silver deposits in relation to the silver bromide crystals from which they derive was investigated. The emulsion gelatin surrounding the crystals was stained whereas the spaces, which remained after the crystals were dissolved in the photographic fixer, appeared transparent. This analysis permitted the selection of development procedures in which the single or multiple round silver deposits originating from a single crystal will remain within or on the boundary of this crystal. By this method, quantitation of radioautographic reactions composed of small, round silver deposits was studied by using the uniformly labeled 3H-methacrylate sections as a standard source of radiation. The conditions under which grain counting is feasible are discussed.     

Histochemical studies of uric acid in some insects. 3. Excretion of uric acid by the malpighian tubules in Calliphora vicina and Rhodnius prolixus

In adult Calliphora uric acid is excreted throughout the Malpighian tubules. Histochemical preparations for the light microscope show uric acid passing through the cells and forming crystalline spheres in immediate contact with the microvilli. Uric acid appears to be synthesized and discharged into the haemolymph by the fat body cells. In Rhodnius there is no visible uric acid in the cells or lumen of the upper segment of the tubule (two-thirds of the total length of the tubule) apart from occasional deposits in the basal lamina. All uric acid excretion depends on the lower segment. Electron micrographs after argentaffin staining show high concentration of uric acid in the cytoplasm below the basal lamina (which also contains uric acid deposits). Uric acid is visible throughout the cell, particularly aroand the mitochondria; it is absent from the infolded plasma membrane and from all vacuoles. At the lumen there is a concentrated deposit of uric acid immediately beyond the plasma membrane. The uric acid particles unite with particles of unstained matrix material to form crystalline spheres. The fat body shows active synthesis of uric acid which is discharged by the cells into the intercellular channels and so to the basal lamina through which it passes into the haemolymph. As judged by histochemical preparations the haemolymph contains a high concentration of uric acid, very variable in different sites. Likewise large variations in uric acid secretion occur in different parts of the fat body.     

Histochemical studies of uric acid in some insects. 2. Uric acid and polyphenols in the fat body

It is recalled that in the larva of Aedes aegypti, starved after a rich protein diet, uric acid is formed and accumulates in the fat body, not as solid spheres but in high concentration in aqueous vacuoles. In the mature larva of Celliphora vicina which has finished feeding and is settling down to form the puparium, the fat body at first contains no argentaffin deposits. During the following 2 or 3 days, argentaffin material appears in the form of amber or brown vesicles and black granules of all sizes. Some of this material remains in the fat body cells; but a large part, presumably polyphenols, is discharged from the cells so that finally all the amber staining disappears and only black granules remain. During this transfer the epidermal cells become charged with sclerotin precursors, which are transferred into the outer part of the cuticle to form the puparium. The stored uric acid remains in the fat body and is dispersed during adult development and ultimately excreted.     

痛风性关节炎动物模型的研究现状与展望

痛风是由于机体嘌呤代谢紊乱,导致血内尿酸增高和/或肾脏排泄尿酸减少,从而引起尿酸盐在组织沉积的疾病,目前尚未见在实验动物中复制出类似人类的痛风性关节炎模型。通过对目前国内外高尿酸血症及痛风模型复制的方法、机制和应用的研究,分析各自的特点及不足之处,并提出复制更加符合临床的高尿酸血症及痛风性关节炎动物模型的展望与设想。     

Purine metabolism in high and low uric acid lines of chickens: de novo uric acid synthesis in isolated hepatocytes and phosphoribosylpyrophosphate amidotransferase activities

The de novo biosynthesis of uric acid was examined in isolated hepatocytes from the high and low uric acid lines of chickens. Rates of incorporation of radiolabeled glycine into uric acid by hepatocytes from the high uric acid (HUA) line were approximately 3.6-fold greater than found in low uric acid (LUA) control hepatocytes. Uric acid synthesis rates in these cells were positively correlated with plasma uric acid levels (r = +0.77; P less than 0.01). The activity of phosphoribosylpyrophosphate (PRPP) amidotransferase was measured in acetone powder preparations from liver and kidney tissues of the HUA and LUA lines. Activities in kidney tissues were about 21% lower than those found in livers. PRPP amidotransferase activities in liver and kidney tissues did not correlate significantly with plasma uric acid levels. The increased synthesis of uric acid in the HUA line may be the result of the increased PRPP synthetase activities and PRPP pool sizes previously reported for these tissues.     

Uric acid-degrading bacteria in the gut of the invading apple snail Pomacea canaliculata and their possible symbiotic significance

In previous studies we have mapped uric acid deposits in specialized tissues of the invading apple snail Pomacea canaliculata. Uric acid stores function as reservoirs of combined nitrogen in insects. The first step for recycling uric acid nitrogen involves the action of uricase and here we explored the occurrence of uricase-positive bacteria in the gut of this snail. Six bacterial strains assigned to the genera Pseudomonas, Enterobacter, Citrobacter and Lactococcus were isolated on the basis of their ability to grow in vitro in a medium containing uric acid as the only carbon and nitrogen source. Uricase specific activity could be determined in three of these genera (not in Citrobacter), showing optimal pH values ranging 7.3–8.7 and optimal substrate concentrations (ranging 25–30 μM). These uricolytic bacteria may participate in recycling of combined nitrogen in this snail.     

Theobromine Inhibits Uric Acid Crystallization. A Potential Application in the Treatment of Uric Acid Nephrolithiasis

Purpose

To assess the capacity of methylxanthines (caffeine, theophylline, theobromine and paraxanthine) to inhibit uric acid crystallization, and to evaluate their potential application in the treatment of uric acid nephrolithiasis.

Materials and Methods

The ability of methylxathines to inhibit uric acid nucleation was assayed turbidimetrically. Crystal morphology and its modification due to the effect of theobromine were evaluated by scanning electron microscopy (SEM). The ability of theobromine to inhibit uric acid crystal growth on calculi fragments resulting from extracorporeal shock wave lithotripsy (ESWL) was evaluated using a flow system.

Results

The turbidimetric assay showed that among the studied methylxanthines, theobromine could markedly inhibit uric acid nucleation. SEM images showed that the presence of theobromine resulted in thinner uric acid crystals. Furthermore, in a flow system theobromine blocked the regrowth of post-ESWL uric acid calculi fragments.

Conclusions

Theobromine, a natural dimethylxanthine present in high amounts in cocoa, acts as an inhibitor of nucleation and crystal growth of uric acid. Therefore, theobromine may be clinically useful in the treatment of uric acid nephrolithiasis.     

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.     

Silver Staining for Nucleolar Organizing Regions of Vertebrate Chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Silver staining for nucleolar organizing regions of vertebrate chromosomes

Refinements to a simple, one-step silver staining technique for nucleolar organizing regions are described. These include fixation of silver stained material with sodium thiosulfate and standardization of silver development conditions for different groups of vertebrates. The central advantages to the method are that it is rapid, reliable, simple, and inexpensive. Additional benefits include (i) consistent and uniform silver staining of nucleolar organizing regions, (ii) few reduced silver deposits elsewhere on the chromosomes or on the slides, (iii) generally unaltered chromosome morphology after silver treatment, and (iv) relative permanence of Permounted preparations. The method works equally well on chromosomes made from cell cultures and from solid tissues of live specimens.     

Reprecipitation during the preparation of demineralized sections. II. Experimental observations.

Teeth were incompletely demineralized by immersion in unchanged 10% formic acid for 7 days. Reprecipitation deposits of secondary calcium phosphate were present in the dentin and soft tissues of the dental pulp and, if the final pH was 3 or greater, in the remnants of the periodontal ligament. The deposits in the dentin appeared to be intratubular. Deminieralized sections of teeth suspended in supersaturated solutions of brushite contained similar deposits in the soft tissues. It is suggested that reprecipitation of secondary calcium phosphates is a frequent intermediate stage during demineralization with formic acid.     

Simultaneous determination of the amounts of metallic and "reducible" silver in histologic specimens.

Acids and weak complexing agents (pK less than 8) are not able to remove, without leaving a residue, silver bound to biological tissues by ionic or complex bonds ("reducible" silver), whereas, strong complexing agents (pK greater than 8) can also partially or completely dissolve metallic silver formed under the influence of reducing groups in the tissue. For this reason, the chemical nature of the silver contained in tissue sections, be it metallic or reducible, must not be determined on the basis of solubility tests; moreover, the amount of neither of the two above fractions can be determined by removing the other with any kind of washing. Using radioactive impregnating baths, radioactive silver bound to the tissue as reducible silver can be replaced in a quantitative manner with inactive silver ions by means of a one-hour incubation in 1% inactive silver nitrate dissolved in 10% acetic acid, but the radioactive silver existing in reduced (atomic) state will be left unaffected. Consequently, radioactivity remaining in the tissue after the above treatment represents metallic silver. The amount of reducible silver can be calculated by subtracting that of the metallic silver from the total silver content of the sections.     

A silver method for counterstaining plastic embedded tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

A Silver Method for Counterstaining Plastic Embedded Tissue

This report presents a method which can be used for counterstaining semithin sections of plastic embedded tissue. The sections are treated with a solution of silver lactate, followed by physical development. During the silver lactate treatment, silver ions are bound by various tissue components as metallic silver or silver sulfide. During physical development catalytic reduction of silver ions to metallic silver takes place where silver has been bound in the tissue, enlarging the silver deposits to microscopically visible dimensions. The amplified silver deposits give high contrast staining in yellow, brown and black suitable for both color and monochrome photography. The localization of the silver deposits is highly specific and may reflect several independent chemical processes. Examples in several tissues are shown.     

Subcellular localization of uric acid storage in the fat body of Manduca sexta during the larval-pupal transformation

The fat body of the tobacco hornworm, Manduca sexta, serves as the major site for uric acid storage during metamorphosis. Light and electron microscopic examinations of fat body stained with reduced silver to show the location of stored uric acid have revealed that most, if not all, fat body cells store uric acid. The extent of specific staining is proportional to the increase in uric acid concentration in fat body during the initial stages of metamorphosis. Storage is associated with discrete membrane-bound structures, designated as uric acid storage vacuoles. In larval fat body, the structures are round or elliptical-shaped vacuoles with electron-dense fibrous interiors and are about the size of observed mitocondria (0.5–1.0 μm). During the larval-pupal transformation, the storage vacuoles double in size and appear as fibrous cores with spaces between the cores and the surrounding membranes. Before pupal ecdysis, the storage vacuoles are concentrated around the nucleus of each cell but after that event they are more uniformly distributed within fat body cells.     

Fluorescence modulation of 1,7‐bis(4‐hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione by silver nanoparticles and its possible analytical application

The effects of silver nanoparticles on the photophysical properties of 1,7‐bis(4‐hydroxy‐3‐methoxyphenyl)‐1,6‐heptadiene‐3,5‐dione, popularly known as curcumin, have been investigated using optical absorption and fluorescence techniques. Although absorption spectroscopy suggests a ground‐state complex formation, fluorescence quenching data confirms a simultaneous static and dynamic quenching, inferring ground as well as excited‐state complex formation. The recovery of fluorescence quenching of the curcumin–silver nanoparticle complex in the presence of ascorbic acid or uric acid emphasizes a strong interaction between the silver nanoparticles and ascorbic acid/uric acid, suggesting that fluorescence recovery after the quenching of curcumin–silver nanoparticle complexes has potential for ascorbic acid or uric acid assay development. Copyright © 2011 John Wiley & Sons, Ltd.     

Is alcoholic acidic silver nitrate reagent really specific for the histochemical localization of ascorbic acid.

The histochemical localization of ascorbic acid in plant tissues with the alcoholic acidic silver nitrate reagent is shown here to be not specific for ascorbic acid, since some of the polyphenolic substances, including flavonoids, which are known to be widely distributed in plant tissues, are also able to reduce the acidic alcoholic silver nitrate reagent at low temperature (0-4 degrees C) and at pH 2 to 2.5 in dark. This method may perhaps be used for animal tissues where flavonoid pigments do not occur in such large quantities as they do in plants. I therefore, come to the inevitable conclusion that the use of alcoholic acidic silver nitrate reagent in localizing ascorbic acid in plant tissues may be highly misleading.     

Regional heterogeneities in the production of uric acid from adenosine in the bivascularly perfused rat liver

The heterogeneity of the liver parenchyma in relation to uric acid production from adenosine was investigated using the bivascularly perfused rat liver in the anterograde and retrograde modes. Adenosine was infused in livers from fed rats during 20 min at four different concentrations (20, 50, 100 and 200 M) according to four experimental protocols as follows: (A) anterograde perfusion, with adenosine infusion into the portal vein; (B) anterograde perfusion, with adenosine in the hepatic artery, (C) retrograde perfusion, with adenosine in the hepatic vein; (D) retrograde perfusion, with adenosine in the hepatic artery. With protocols A, B, and D uric acid production from adenosine was always characterized by initial bursts followed by progressive decreases toward smaller steady-states. With protocol C the initial burst was present only when 200 M adenosine was infused. The initial bursts in uric acid production were accompanied by simultaneous increases in the ratio of uric acid production/adenosine uptake rate. These initial bursts are thus representing increments in the production of uric acid that are not corresponded by similar increments in the metabolic uptake rates of adenosine. Global analysis of uric acid production revealed that the final steady-state rates were approximately equal for all infusion rates with protocols A, B and C, but smaller with protocol D. This difference, however, can be explained in terms of the differences in accessible cellular spaces, which are much smaller when protocol D is employed. When the analysis was performed in terms of the extra amounts of uric acid produced during the infusion of adenosine, where the initial bursts are also taken into account, different dose-response curves were found for each experimental protocol. These differences cannot be explained in terms of the accessible cell spaces and they are likely to reflect regional heterogeneities. From the various dose-response curves and from the known characteristics of the microcirculation of the rat liver it can be concluded that the initial bursts in uric acid production are generated in periportal hepatocytes. The reason for this heterogeneity could be related to the metabolic effects of adenosine, especially to oxygen uptake inhibition, which is likely to produce changes in the ATP/AMP ratios.     

Postmortem changes of uric acid in various rat tissues: Determination of uric acid by reversed-phase high-performance liquid chromatography with electrochemical detection

As a function of time after decapitation, postmortem changes of uric acid in various rat tissues have been studied by reversed-phase high-performance liquid chromatography with electrochemical detection. The chromatographic examination revealed that uric acid in rat tissues such as brain, liver, musculus rectus abdominis, and femoral muscle tends to increase after decapitation as a function of time between the sacrifice and homogenization in a 2.0% metaphosphoric acid solution.     

Uric Acid Secretion from Adipose Tissue and Its Increase in Obesity

Obesity is often accompanied by hyperuricemia. However, purine metabolism in various tissues, especially regarding uric acid production, has not been fully elucidated. Here we report, using mouse models, that adipose tissue could produce and secrete uric acid through xanthine oxidoreductase (XOR) and that the production was enhanced in obesity. Plasma uric acid was elevated in obese mice and attenuated by administration of the XOR inhibitor febuxostat. Adipose tissue was one of major organs that had abundant expression and activities of XOR, and adipose tissues in obese mice had higher XOR activities than those in control mice. 3T3-L1 and mouse primary mature adipocytes produced and secreted uric acid into culture medium. The secretion was inhibited by febuxostat in a dose-dependent manner or by gene knockdown of XOR. Surgical ischemia in adipose tissue increased local uric acid production and secretion via XOR, with a subsequent increase in circulating uric acid levels. Uric acid secretion from whole adipose tissue was increased in obese mice, and uric acid secretion from 3T3-L1 adipocytes was increased under hypoxia. Our results suggest that purine catabolism in adipose tissue could be enhanced in obesity.