The preservation of ultrastructure in saturated phosphatidyl cholines by tannic acid in model systems and type II pneumocytes

The preservation for electron microscopy of saturated phospholipids in general, and phosphatidyl choline (PC)in particular, remains and unsolved problem since OsO(4) and glutaraldehyde are incapable of interacting with PC directly. However, by introducing tannic acid preceding osmication, we were able to demonstrate highly ordered, preserved lamellar structures in model experiments with saturated PC, and in vivo experiments type II pneumocytes of lung tissue. The secretory bodies of the latter are known to contain a high proportion of these saturated phospholipids. In both cases, the repeating periodicity approximated 45 A. It was determined that tannic acid interacts with the choline component of PC to form a "complex," which then could be stabilized by treatment with OsO(4). In the absence of osmication, the PC-tannic acid complex acid did not survive conventional dehydration techniques, but osmication permitted conventional Epon embedment. Sphingomyelin (SPH), which contains choline, behaved similarly in model experiments. But there was no evidence of a comparable reaction with tannic acid using phosphatidyl ethanolamine (PEA), phosphatidyl serine (PS), or phosphstidy inositol (PI). Chemical studies indicted a high pH dependency for the formation of the PC- tannic acid complex. Also, experiments demonstrated its dissociation in various organic solvents. Sharp delineation and great contrast of the polar zones in the ordered lamellar structures was achieved by additional staining with lead citrate thus leading to the conclusion that tannic acid serves as a multivalent agent, capable of simultaneous interaction with saturated PC, OsO(4), and lead citrate stains.     

Staining of the elastic fibers in insect connective tissue after tannic acid/glutaraldehyde fixation.

Locust neural lamella and Calpodes connective tissue fixed in glutaraldehyde have a fibrous component which stains after reaction with DAB and osmication and after staining sections with PTA. The fibers also stain when fixed in glutaraldehyde with tannic acid followed by osmication and section staining with lead citrate.     

Ultrastructural visualization of elastic fibres with a tannate-metal salt method

Summary A modification of the tannic acid-metal salt method was applied as an ultrastructural stain for elastin. Thin sections of glutaraldehyde-fixed, embedded rat aorta and rabbit elastic cartilage, with and without osmication, were examined. Raising the pH of the tannic acid solution from 2.7 to 9.0 progressively increased the electron-density of elastic fibres and collagen fibrils in osmicated and unosmicated specimens. The maximum tannic acid staining of elastic fibres was observed in the pH range 7.0–9.0. Collagen staining, although less intense than that of elastic fibres, was also greatest in this pH range. Elastic fibres in osmicated specimens demonstrated the strongest tannic acid staining with a minimal increase in density of collagen and cell nuclei when compared to the unosmicated specimens. Sequential treatments of osmicated specimens with tannic acid pH 7.0–9.0, and uranyl acetate, pH 4.1, enhanced the density of the elastin intensely, increased collagen staining moderately, but hardly increased the density of nuclei and microfibrils. In elastase-digested osmicated specimens, all tannic acid (pH 7.0)-uranyl acetate-reactive elastin was selectively removed. These results demonstrate that all the neutral and alkaline tannic acid-uranyl acetate methods can be used as a postembedment stain for elastin specimens fixed in glutaraldehyde and osmium tetroxide.     

The use of tannic acid for the ultrastructural visualization of hyaluronic acid

Summary This study describes a method, which makes use of tannic acid (2%) as a component of a paraformaldehyde-glutaraldehyde based fixative, to reveal the presence and ultrastructure of glycosaminoglycans in the extracellular matrix. The ultrastructure of the extracellular matrix in the stage 24 chick embryo wing is examined after fixation by several procedures. After fixation in the absence of tannic acid, the intercellular spaces contain little extracellular matrix, except for occasional fibrils (collagen?). On the other hand, when tannic acid is included in the primary fixative, the intercellular spaces contain considerable amounts extracellular matrix which includes 3±0.5 nm filaments, ±30 nm granules, as well as putative collagen fibrils. The 3±0.5 nm diameter fibrils are not observed when the limbs had been injected in ovo with Streptomyces hyaluronidase (specific for hyaluronic acid) prior to fixation. Furthermore, the 3±0.5 nm fibrils resemble authentic hyaluronic acid that had been fixed by the same procedure in the presence of tannic acid. Limbs treated with tannic acid after osmication contained only small amounts of extracellular material, which was confined largely to cell surfaces. These results demonstrate that the use of tannic acid in the primary fixative can serve as a useful method for the ultrastructural visualization of several extracellular matrix materials, including hyaluronic acid.This study was supported by NIH grant HD 05505     

Phalloidin-eosin followed by photo-oxidation: a novel method for localizing F-actin at the light and electron microscopic levels.

We describe a novel high-resolution method to detect F-actin at the light and electron microscopic levels through the use of the actin-binding protein phalloidin conjugated to the fluorophore eosin, followed by photo-oxidation of diaminobenzidine. This method possesses several key advantages over antibody-based labeling and structural methods. First, phalloidin binding to F-actin can tolerate relatively high concentrations of glutaraldehyde (up to 1%) in the primary fixative, resulting in good ultrastructural preservation. Second, because both eosin and phalloidin are relatively small molecules, considerable penetration of reagents into aldehyde-fixed tissue was obtained without any permeabilization steps, allowing 3D reconstructions at the electron microscopic level. By employing a secondary fixation with tannic acid combined with low pH osmication, conditions known to stabilize actin filaments during preparation for electron microscopy, we were able to visualize individual actin filaments in some structures. Finally, we show that fluorescent phalloidin can be directly injected into neurons to label actin-rich structures such as dendritic spines. These results suggest that the fluorescent phalloidin is an excellent tool for the study of actin networks at high resolution.     

Studies on interactions between plant secondary metabolites and glutathione transferase using fluorescence quenching method

The interactions between plant secondary metabolites (tannic acid, rutin, cinnamic acid and catechin) and glutathione transferase (GST) were investigated by fluorescence and UV-Vis absorption spectroscopy. Intrinsic fluorescence of GST was measured by selectively exciting their tryptophan (Trp) residues and quenching constants were determined using the Stern-Volmer equation. The binding affinity was found to be strongest for tannic acid and ranked in the order tannic acid>rutin>cinnamic acid>catechin. The pH values in the range of 6.7-7.9, except for tannic acid, did not affect significantly the affinity of rutin, cinnamic acid and catechin with GST. Results showed that the fluorescence quenching of GST was a static_quenching. Fluorescence quenching and UV-Vis absorption spectroscopy suggested that only the tannic acid changed the microenvironment of the Trp residues. Furthermore, the number of binding sites and binding constants at different pH values showed that tannic acid had strongest affinity towards GST and hydrogen bonding played an important role in the affinity between GST and the metabolites.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase

Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.     

单宁酸对根田鼠断乳幼体生长和存活的影响

在食物中含10％和20％蛋白质条件下,测定了单宁酸对根田鼠幼体在断乳后20d内的生长和存活的影响。结果表明,食物中含蛋白质为10％的条件下,摄食含3％和6％单宁酸食物的试验个体生长速率分别为-0．135g/d和-0．25g/d,食物利用效率均显低于对照组,断乳后20d内平均存活天数较对照组分别下降26．23％和49．36％。在食物蛋白质为20％的条件下,摄食含3％和6％单宁酸食物的试验个体生长速率分别为0．134g/d和-0．116g／d,摄食6％单宁酸食物的试验个体食物利用效率显低于摄食3％单宁酸食物的试验个体和对照组个体,断乳后20d内的平均存活天数较对照组下降39．41％,摄食3％单宁酸食物的试验个体较对照组略有下降,但不显。上述结果验证了单宁酸能显影响植食性小哺乳动物生长和存活的假设。     

Differential fixation of poly(L-arginine) and poly(L-lysine) by tannic acid and its application to the fixation of collagen in electron microscopy

A differential fixation of poly(L-arginine) and poly(L-lysine) has been demonstrated by means of cellulose acetate electrophoresis and colorimetric titration. Electrophoresis showed that at pH 3.0 and concentrations between 0.025% and 2% the reagent interacts with poly(L-arginine) but not with poly(L-lysine). at pH 7.5, however, poly(L-lysine) also reacts, although at a higher concentration of tannic acid than was required to fix poly(L-arginine) at this pH. Colorimetric titration revealed that for poly(L-arginine) the reaction with tannic acid commences at pH 3.0 and is complete at pH 4.1 whereas for poly(L-lysine) the reaction commences at pH 3.5 and is complete at pH 4.9. It is suggested that the reaction is predominantly electrostatic. The results are discussed in relation to the use of tannic acid as a protein fixative in electron microscopy.     

The effects of simultaneous variation in protein, digestible carbohydrate and tannic acid on the feeding behaviour of larval Locusta migratoria (L.) and Schistocerca gregaria (Forskal). I. Short-term studies

ABSTRACT The influence of simultaneously varying the levels in artificial diets of protein, digestible carbohydrate (14% or 28%) and tannic acid (absent or 10%) on the feeding behaviour of the oligophagous Locusta migratoria (L.) and the polyphagous Schistocerca gregaria (Forskal) (Acrididae) was investigated. Total consumption and detailed feeding behaviour were recorded over a 12 h period in choice and no-choice experiments. In addition, amounts eaten by Schistocerca of the 14% protein, 14% carbohydrate diet with and without tannic acid were measured at regular intervals throughout the fifth stadium, and insect growth over this period was recorded. There were no interactive effects of nutrient levels and tannic acid, despite the fact that both species compensated for dilution of dietary protein by increasing consumption. Only male Locusta compensated for dilution of dietary carbohydrates, and this compensation was much less marked than for protein. Tannic acid did influence feeding as a main effect, however. It caused an increase in amounts eaten by Schistocerca in both choice and no-choice experiments. This increased consumption was due to an increase in the number of meals taken. A shorter latency period before and a longer duration of the first meal by naive insects suggested a phagostimulatory rather than a post-ingestive effect of tannic acid. The stimulatory effect was only apparent for the first 24 h of continuous exposure, but this temporary enhancement none the less resulted in the insects being heavier at adult ecdysis. Stadium duration was also somewhat reduced. In a no-choice situation, no effect of tannic acid on the feeding behaviour of Locusta was observed. When given a choice, however, this species took significantly more meals on the tannic acid-free diet, these being of similar average size to meals taken on the tannic acid diet. Significantly more insects took their first meals on the tannic acid-free diet in the choice test, indicating a deterrent effect of tannic acid in Locusta.     

Enhanced ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine reaction product

Ultrastructural visualization of the horseradish peroxidase-tetramethylbenzidine (HRP-TMB) reaction product within trigeminal ganglion cells and brain stem axons and terminals following HRP injections into the pulpal chambers of cat teeth is enhanced by utilization of a modified osmication procedure that converts the reaction product to a markedly stable and electron-dense form. The results following the use of the modified osmication procedure (pH 5.0 phosphate buffer at 20 degrees C for 12 hours) are compared to results obtained by following Carson's osmication protocol (Carson KA, Mesulam M-M: J Histochem Cytochem 30:425, 1982; Carson KA, Mesulam M-M: In Tracing Neural Connections with Horseradish Peroxidase. Edited by M-M Mesulam. J Wiley, Chichester, England, 1982, p 153-184) (pH 6.0 phosphate buffer at 45 degrees C for 45 min). The results suggest that the conversion of the HRP-TMB reaction product to an electron-dense form during osmication is intimately associated with the pH of the phosphate buffer and the total time of osmication.     

A labile,Ca2+-dependent cytoskeleton in rhabdomeral microvilli of blowflies

Summary Rhabdomeral microvilli of photoreceptors of the blowfly Lucilia are shown to contain a cytoskeleton. An axial filament ( 6–11 nm) in each microvillus is inserted into a terminal cap distally, and into a plug filling the narrow neck of the microvillus proximally. In some states, the axial filament projects beyond the neck; within the microvillus it is surrounded by amorphous material. Together, they form an axial complex, which supports side-arms linking it to the plasma membrane. Conventional fixation for examination with the electron microscope destroys the cytoskeleton. To preserve it, retinae are pre-treated with a Ringer's solution buffered with 20 mM imidazole and containing, minimally, the following components: (i) a protease inhibitor, usually phenylmethylsulphonyl-fluoride (PMSF); (ii) either the Ca²⁺-chelator EGTA, or the calmodulin-blocking agent trifluoperazine (TFP); and (iii) a source of divalent cations to preserve the side-arms. When EGTA is used, Mg²⁺, Sr²⁺, Ba²⁺, Mn²⁺ and Co²⁺ are effective, Ba²⁺ giving the most satisfactory contrast, and Mg²⁺ and Co²⁺ the best preservation. It is inferred that the cytoskeletal complex includes at least one Ca²⁺-activated protease, and possibly calmodulin. Microvilli are bonded together by intermicrovillar bridges with a periodicity of 11–17nm. The cytoskeleton is destroyed by pretreatment with 1 mM dithiothreitol (DTT), possibly by the activation of a thiol protease. It does not survive osmication unless treated with low molecular weight tannic acid (LMWT). The evidence does not discriminate between actin and intermediate filaments as the basis of the cytoskeleton. Attention is drawn to similarities and differences between the rhabdomeral cytoskeleton and that of vertebrate intestinal brush-borders. The extreme lability of the rhabdomeral cytoskeleton to conventional methods of fixation is attributed in part to the Ca²⁺ fluxes experienced by invertebrate photoreceptors, and in part to the effects of osmication.The authors thank Dr. Lindsay Barton-Brown and Tom Van Gerwen for supplying flies from CSIRO cultures: Smith Kline and French Laboratories Ltd., French's Forest, N.S.W. for a generous gift of trifluoperazine, and Mallinckrodt, Inc., St. Louis, Missouri, USA, for a gift of low molecular weight tannic acid. Many colleagues, especially Richard Payne, Steve Shaw and Gert De Couet helped by discussing the results. George Weston and the staff of the Electron Microscope Unit provided support and advice. Sandy Smith prepared Table 1     

Captive roe deer (Capreolus capreolus) select for low amounts of tannic acid but not quebracho: fluctuation of preferences and potential benefits

Browsing ruminants have been shown to tolerate a certain amount of tannins in their natural diet, and preference trials with captive roe deer (Capreolus capreolus) have suggested an active selection for a low dose of hydrolysable tannins. In this study, we investigated the preference patterns for tannic acid, a source of hydrolysable tannins, and quebracho, a source of condensed tannins, in a series of preference trials with captive roe deer over time, using a pelleted feed that differed only in the respective tannin content. Additionally, two groups of four hand-raised roe deer fawns were fed either a control or a 3% tannic-acid containing diet and physiological parameters were compared after 7.5 months. There were large differences in preference patterns between the individual roe deer groups; quebracho was mostly avoided, whereas tannic acid was actively included in the diet in differing, low proportions. However, one group consistently preferred the quebracho diet over both the control or the tannic acid diet. For the tannic acid, the preference pattern often revealed an initial period of high preference, followed by a stable period of a moderate preference. The fawns on the tannic acid diet had a lower pellet intake and a higher relative mass gain than the fawns on the control diet; differences in salivary tannin-binding capacity and in blood antioxidant status were below significance. These results are the first indications of potential benefits of a low-dose tannin diet, which need further confirmation. The results of the preference trials demonstrate that the time pattern of tannin intake is not constant, and pose the question about the validity of short-term preference trials in general.     

单宁酸对淡色库蚊抗氰戊菊酯品系和敏感品系幼虫生长发育的影响

单宁酸是一种植物次生代谢物, 为探索其用于蚊幼虫防治的可能性,在室内测定了其对淡色库蚊Culex pipiens pallens抗氰戊菊酯品系和敏感品系1～4龄幼虫的毒性,并观察了其对存活幼虫生长发育的影响。结果表明,淡色库蚊敏感品系幼虫对单宁酸的敏感性比抗氰戊菊酯品系的要高,1～4龄幼虫分别高6.4、4.9、4.7和2.0倍。4个龄期幼虫中,无论是敏感品系还是抗氰戊菊酯品系,均是1龄幼虫对单宁酸的敏感性最高,3龄幼虫最低。在1 000 mg/L单宁酸持续作用下,敏感品系和抗氰戊菊酯品系各龄幼虫的存活率,均随处理时间延长而降低。与对照相比,饲养在100 mg/L～500 mg/L单宁酸溶液中的存活幼虫发育历期延长,敏感品系和抗氰戊菊酯品系发育历期分别延长了34.5～38.3 h和59.2～93.4 h。其中,125 mg/L浓度处理的敏感品系1～4龄幼虫,其发育历期与对照的差异达到了显著水平(P<0.05);抗性品系则在250 mg/L作用下也达到了差异显著水平(P<0.05)。但100～250 mg/L单宁酸处理淡色库蚊抗氰戊菊酯品系和敏感品系1龄幼虫,对其存活幼虫的化蛹率、羽化率和成虫性比均无显著影响。表明单宁酸对淡色库蚊幼虫的影响主要是延迟其生长发育,且影响程度与蚊虫对氰戊菊酯的敏感性有关。     

Dual effect of tannic acid on the preservation and ultrastructure of phosphatidyl choline vesicles

Summary The use of tannic acid has been proposed to improve the preservation of phospholipids in tissues. We investigated the effects of tannic acid on the preservation of small unilamellar vesicles, prepared from sonicated aqueous suspensions of phospholipids.With cryo-electron microscopy it is demonstrated that small unilamellar vesicles are formed after sonication of the phospholipid suspensions. Fixation of vesicles without tannic acid results in extraction of the phospholipids during dehydration and embedding. Fixation of vesicles containing phosphatidyl choline with tannic acid, with or without glutaraldehyde, results in a fast (within a second) aggregation of the vesicles and the resulting sediment can be dehydrated and embedded when a postfixation in osmium tetroxide is carried out. Small unilamellar vesicles fixed in this way are retrieved in thin sections as multilamellar vesicles with a periodicity of about 5 nm for dimyristoylphosphatidyl choline and about 6 nm for dioleoylphosphatidyl choline.By using ¹³C-phosphatidyl choline it was also demonstrated that tannic acid prevents to a large extend the extraction of phosphatidyl choline during fixation, dehydration and embedding. This dual effect of tannic acid on phosphatidyl choline, aggregation and fixation, should be considered when using tannic acid in tissue preparation.     

Tannic acid in histology: an historical perspective

The development of tannic acid as a reagent in histological methods is traced against a background of widespread use in science and technology from times of antiquity. Numerous light microscopic methods involving tannic acid, particularly in conjunction with iron and silver, have been described for a variety of tissue components. In most applications, tannic acid functions as a mordant. Current use is generally restricted to methods based on its affinity for collagen. The most significant histological use of tannic acid in contemporary times is as an adjunct to conventional glutaraldehyde-osmium-heavy metal fixation and staining for ultrastructural studies of tissue structures not normally clearly demonstrated. Tannic acid reacts with various components by mechanisms which are often not fully understood.     

A three-dimensional study of organelle interrelationships in regenerating rat liver. 3. Organelles related to bile.

A number of cell structures are described which show a morphological relationship to the bile canaliculi. Two types of peribiliary vesicles are identified: osmication positive ones occurring between the bile canaliculi and the osmicated immature Golgi cisternae and probably deriving from the latter, and osmication negative ones related to MVB, on which they appear as buds. Small coated vesicles are seen attached to this second type. Large lacunae may originate from MVB, as suggested by the MVB-like internal vesicles they may contain. Some stay in luminal continuity with the bile canaliculi. Canalicular coated vesicles are seen as parts of the canalicular plasma membrane and free in the cytoplasm.     

Tannase activity from the marine cyanobacterium Phormidium valderianum BDU140441

The marine cyanobacterium Phormidium valderianum BDU 140441 exhibited the ability to grow at 0.25?mM tannic acid, a known hindering chemical for microbial growth. The tannic acid-degrading ability of the organism is evident from the UV–visible absorption spectrum. In addition, the existence of tannase has been localized by activity staining, and its induction in activity upon tannic acid exposure was confirmed in native gel. The critical tannic acid metabolization enzymes tested for are polyphenol oxidase and esterases; both are well known for tannic acid degradation. Upon tannic acid exposure, increased activity of polyphenol oxidase and expression of few new isoforms of esterase were identified by activity staining.     

Differential effects of tannic acid on cisplatin induced nephrotoxicity in rats

Cisplatin is a widely used antineoplastic drug. Major drawback of cisplatin therapy is its nephrotoxicity. The objective of this study was to check the effect of tannic acid on cisplatin induced nephrotoxicity. Post-treatment of tannic acid prevents cisplatin (5mg/kg) induced nephrotoxicity and decreases poly(ADP-ribose) polymerase cleavage, phosphorylation of p38 and hypoacetylation of histone H4. In contrast, co-treatment of tannic acid potentiates the nephrotoxicity. Comparative nephrotoxicity studies show that co-treatment of tannic acid with reduced dose of cisplatin (1.5mg/kg) developed almost similar nephrotoxicity. MALDI protein profiling of plasma samples provides indirect evidence that tannic acid co-treatment increases bioavailability of cisplatin.