Ultrastructural demonstration of exocytosis of neural, neuroendocrine and endocrine secretions with an in vitro tannic acid (TARI-) method

The release of neural, neuroendocrine, and endocrine secretory products by exocytosis was ultrastructurally studied by means of tissue incubation in Ringer containing tannic acid (Tannic Acid Ringer Incubation-method; TARI -method), followed by conventional fixation. Tannic acid strongly enhances the electron density of extracellular (secretory) substances. During TARI -treatment of tissues exocytosis proceeds, but the exteriorized contents of the secretory granules are immediately fixed by tannic acid and do not diffuse away into the extracellular space. In this way detection of exocytosis is markedly facilitated since the number of exocytosis phenomena visible at the ultrastructural level considerably increases with progressing incubation time. Studies of the central nervous system of the mollusc Lymnaea stagnalis show that the occurrence of exocytosis during TARI -treatment is calcium-dependent. With the TARI -method exocytosis has been clearly demonstrated in a variety of structures (endocrine cells, neurohaemal axon terminals, synapses) of L. stagnalis, the insect Locusta migratoria, and the rat, including cell types where exocytotic release had not been shown before.     

Ultrastructural demonstration of exocytosis in intact and saponin-permeabilized cultured bovine chromaffin cells

Exocytosis is the release of intracellular vesicular contents directly to the cell exterior after fusion of the vesicular and plasma membranes. It is generally accepted as the process by which transmitters and hormones are released from neurons and neurosecretory cells. There is overwhelming biochemical evidence that exocytosis is the mechanism by which catecholamines are released from adrenal chromaffin cells. With the exception of the hamster, however, there is little ultrastructural evidence to support such a mechanism. We have used a modified in vitro tannic-acid method to visualize exocytosis by transmission electron microscopy in intact and saponin-permeabilized bovine chromaffin cells. When cells are exposed to tannic-acid-containing medium, the content of vesicles involved in exocytosis is coagulated in situ as the vesicle opens to the exterior. Numerous exocytotic profiles were observed. The exposed vesicle contents appeared more granular than those of vesicles in the cell interior. Tannic acid also made the plasma membrane more distinct. Small holes were apparent in the plasma membrane of saponin-treated cells, with little disruption of underlying cytoplasmic structure. Furthermore, when these cells were stimulated with calcium, exocytosis was evident only at regions of intact plasma membrane, not at the holes. Parallel measurements of secretion showed no secretion in the presence of tannic acid. Pretreatment with tannic acid prevented subsequent secretion by intact cells and markedly reduced that of permeabilized cells, indicating a probable change in the nature of the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)     

根田鼠对食物单宁酸的解毒代价

在实验室条件下,测定了根田鼠对单宁酸的解毒代价。结果表明,在食物蛋白质为10％的条件下,摄食3％和6％单宁酸食物的根田鼠经尿液分泌的葡萄糖醛酸较对照组分别增加13．77％和38．80％;与对照组比较,在食物蛋白质为20％的条件下,摄食3％和6％单宁酸食物的实验个体尿液分泌的葡萄糖醛酸分别增加6．11％和22．25％。在食物蛋白质为10％时,用3％和6％单宁酸食物处理的个体,其尿液NH4^ —N含量较对照组个体分别增加51．69％和198．44％。在食物中蛋白质为20％时,摄食3％和6％单宁酸食物的试验个体,其尿液中的NH4^ —N含量分别较对照组高1．72％和74．19％。由此说明,单宁酸能显著增加根田鼠尿液葡萄糖醛酸分泌量,根田鼠尿液的NH4^ —N的含量随食物单宁酸增加而增高,从而增加动物机体对单宁酸的代谢价。     

Enzymatic synthesis of gallic acid from tannic acid with an inducible hydrolase of Enterobacter spp

Gallic acid acts as a precursor molecule to synthesize various tannin molecules. These are plant polyphenols and were proved to be good anti-oxidant, anti-cancerous, anti-inflammatory, anti-microbial compounds. In order to fully exploit prominent biological activities of specific tannins and to develop tannin-based new medicines, it is necessary to obtain their pure preparations with an aim of high yield and specificity. In the present study, gallic acid is synthesized by the hydrolysis of tannic acid using a microbial based transformation process. The microorganism was isolated and identified. The ability of the isolated microorganism to covert tannic acid into gallic acid was determined by HPLC and enzyme production.

• Highlights

• The present investigation signifies the role of Enterobacter spp. in various processes:

• ??To synthesize gallic acid (a precursor for food oxidant such as propyl gallate) and a bacteriostatic antibiotic (trimethoprim).

• ??To protect the environment from tannery’s discharge through the process of biodegradation.

• ??To reduce the toxicity of tannins in animal feed.

     

Ultrastructural cytochemical and autoradiographic demonstration of nonspecific esterase(s) in guinea pig basophils

We used ultrastructural autoradiographic and cytochemical methods to localize esterase activities in unstimulated guinea pig basophils and in basophils undergoing degranulation or recovery from degranulation. We used tritium-labeled diisopropylfluorophosphate (DFP) as a probe for serine enzymes and localized this probe by ultrastructural autoradiography to cytoplasmic granules of immature or mature unstimulated basophils, as well as to granules released by degranulating basophils. Ultrastructural cytochemistry using alpha naphthyl acetate (ANA) as substrate localized nonspecific esterase activity to extruded granules, either within the interiors of degranulation sacs or within granules completely separated from degranulating basophils. Extruded granules retained their esterase activity for as long as 24 hr after antigen-induced degranulation. The plasma membranes of unstimulated or degranulating basophils, as well as of basophils recovering from degranulation, displayed prominent cell surface ANA esterase ectoenzyme activity. Lipid bodies, organelles present in the cytoplasm of both control and recovering basophils, were also alpha naphthyl acetate esterase (ANAE)-positive. Thus, cytochemical and autoradiographic techniques localized esterase and/or [3H]-DFP-binding activities to cytoplasmic granules, lipid bodies, and cell surface of basophils, and these enzyme activities persisted during both degranulation and recovery from degranulation.     

Suppression if milk secretion (exocytosis) by concanavalin A in vitro

The observation that concanavalian A can inhibit milk secretion was evaluated in an in vitro system employing minced mammary gland or isolated alveoli from lactating rats. Release of milk constituents (casein, lactose and fat globules) into the medium in the presence and absence of concanavalin A was measured during 1 or 2 h incubations. The effect of concanavalin A on glucose uptake and CO₂ production of the minced tissued was also studied. Concanavalin A suppressed release of milk components at a concentration as low as 80 μg/ml of medium. Respiration of minced mammary tissue in the presence of concanavalin A (100 μg/ml of medium) was essentially the same as that of the control. The data are evidence that concanavalin A acts directly on the mammary cell in suppressing milk secretion and that the effect is not due to cytotoxicity.     

食物单宁酸对根田鼠(Microtus oeconomus)钠平衡的作用

验证了单宁酸能引起植食性哺乳动物钠平衡的假设.在两种食物蛋白质水平条件下,测定了摄食含0%,3%和6%单宁酸食物的根田鼠的钠平衡和肾上腺肾小球的大小.结果表明,食物单宁酸能显著影响根田鼠的钠平衡,钠的丢失主要通过尿液.同时,单宁酸能显著影响试验个体的肾上腺肾小球的体积.初步研究显示,植食性哺乳动物对摄入的植物次生化合物的解毒引起机体钠平衡失调,而非植物次生化合物的消化抑制作用所致.     

Ultrastructural observations of isolated intact and fragmented junctions of skeletal muscle by use of tannic acid mordanting

Tannic acid mordanting during fixation of isolated vesicles from skeletal muscle enhanced the resolution of the images. Isolated triadic junctions displayed two characteristic features not previously described: (a) a clear gap separated terminal cisternae from transverse tubules; (b) this gap was bridged by a separating array of structures which resembled the "feet" of intact muscle. When the triad was broken in a French press and subsequently reassembled by joining the two organelles, a similar gap was seen but the structure of the feet was less well defined. When the membrane of the triad was extracted by Triton X-100, the junctional region was retained and a similar gap between the two organelles could be discerned. The terminal cisternae characteristically displayed a thickening of the cytoplasmic leaflet of the membrane in select areas in which electron-dense material was apposed on the luminal leaflet. This thickened membrane was not observed in longitudinal reticulum or in terminal cisternae regions distal to the electron-dense matter. This thickened leaflet was not invariably associated with the junction, and some junctional regions did not display discernible thickening of the membrane. When the triad was treated with KCl, the electron-dense aggregate was dispersed and the thickened leaflet of the terminal cisternae dissipated, whereas the triadic junctional region with its feet remained unchanged. KCl treatment caused dissolution of three proteins of Mr = 77,000, 43,000, and 38,000. Treatment of Triton-resistant vesicles with KCl caused the loss of electron-dense aggregate but did not otherwise influence the appearance of the junction. A good degree of correlation both qualitatively and in quantitative parameters between the isolated vesicles and the intact muscle was observed.     

Hamster pulmonary endocrine cells with neural cell adhesion molecule (NCAM) immunostaining

Pulmonary endocrine cells of Syrian golden hamster were stained for neural cell adhesion molecule (NCAM) with indirect fluorescent immunostaining and observed with a confocal laser scanning microscope equipped with an argon laser. Sections 100 m thick of hamster lung fixed with phosphate-buffered 4% paraformaldehyde were prepared. The sections were incubated with rat monoclonal antibody against NCAM, followed by fluorescence-labeled antibody against rat immunoglobulin. Some were doubly immunostained for NCAM and one of the following endocrine markers: neuron-specific enolase, calcitonin gene-related peptide and serotonin. Expression of NCAM in the hamster airway epithelium was seen in cell nests resembling neuroepithelial bodies (NEBs). NCAM immunostaining was positive at the lateral cell borders between the cells composing the nest, but negative at the border with the adjacent, presumably non-endocrine cells. Double immunostaining confirmed that the grouped cells with NCAM immunoreactivity were of an endocrine nature, but that single endocrine cells did not show NCAM immunoreactivity. An electron microscopic study with NCAM immunostaining confirmed the light microscopic study. These suggest that NCAM expression could be important for the morphogenesis of NEBs. A confocal laser microscope was used to make theee-dimensional images of NEBs after NCAM immunostaining and the spatial interaction between NEBs and the surrounding microenvironment was studied.     

Syntaxin requirement for Ca2+-triggered exocytosis in neurons and endocrine cells demonstrated with an engineered neurotoxin

Botulinum neurotoxins cleave synaptic SNAREs and block exocytosis, demonstrating that these proteins function in neurosecretion. However, the function of the SNARE syntaxin remains less clear because no neurotoxin cleaves it selectively. Starting with a botulinum neurotoxin that cleaves both syntaxin and SNAP-25, we engineered a version that retains activity against syntaxin but spares SNAP-25. These mutants block synaptic release in neurons and norepinephrine release in neuroendocrine cells, thus establishing an essential role for syntaxin in Ca2+-triggered exocytosis. These mutants can generate syntaxin-free cells as a useful experimental system for research and may lead to pharmaceuticals that target syntaxin selectively.     

Ultrastructural studies of lizard (Anolis carolinensis) myogenesis in vitro

In vitro differentiation of lizard (Anolis carolinensis) skeletal muscle cells was studied by electron microscopy. Myogenesis was studied under conditions in which large numbers of postmitotic prefusion myoblasts accumulate (Growth Medium) and under conditions which are permissive for myotube formation (Fusion Medium). In Growth Medium, myogenic cells proliferate, then assume a characteristic spherical morphology which permits definitive identification of prefusion myoblasts. During the early stages of culture, these round myoblasts resemble myoblasts described in other systems; ultrastructural similarities and differences are discussed. After longer periods of culture in Growth Medium, a continuum of differentiation from isolated myofilaments to assembled myofibrils was seen in these mononucleated cells. These observations confirm the dissociability of contractile protein assembly and myoblast fusion Cultures maintained in Fusion Medium or transferred from Growth Medium to Fusion Medium form multinucleated myotubes on a predictable time scale. Myogenesis was followed in these cultures with particular reference to the early events in myofilament assembly and myofibril formation.     

Ultrastructural and immunohistochemical study of endocrine cells in the midgut of the cockroach Blaberus craniifer (Insecta,Dictyoptera)

Summary The midgut of Blaberus craniifer is principally made up of columnar epithelial cells which are derived from small regenerative cells found grouped in nidi. Between them, small sparsely granulated cells with clear cytoplasm can be observed lying on the basal lamina. Mainly based on the size, shape and texture of their secretory granules, at least ten types of such endocrine cells have been identified. Five cell types contain a uniform population of dense granules: (1) medium-sized, round to oval granules; (2) small elongated granules; (3) large irregular granules; (4) oval granules with a highly osmiophilic core; (5) oval, haloed granules. Five others are characterized by a heterogeneous population of granules: (6) small, round to oval, variably electron-dense granules; (7) oval medium-sized granules of variable electron density; (8) large irregular granules of variable electron density; (9) small dense granules and large vesicles with filamentous material; (10) small dense granules and very large pale vesicles.In addition, near the regenerative cells, large cells characterized by very large, irregular, dense granules (up to 4 m), lack contact with the lumen, and reach the basal lamina only by slender cytoplasmic processes.Several antisera raised against mammalian peptides and amine were used to reveal axonal fibers and endocrine cells. Serotonin-like immunoreactivity is localized in a profuse innervation of the muscle layers that surround the epithelium, whereas cholecystokinin and methionine-enkephalin antisera stain a more moderate number of axonal fibers. Cholecystokinin-, methionine-enkephalin-, substance P-, vasoactive intestinal peptide-, somatoliberin-, and gonadoliberin-like immunoreactivities were detected in endocrine cells of the epithelium. While most of the cells appear pyramidal, oval, fusiform or bowl-shaped, and seem to lack contact with the lumen, cells reaching it have been detected reacting with antisera to cholecystokinin, substance P, vasoactive intestinal peptide, somatoliberin and gonadoliberin.     

Anti-amyloidogenic activity of tannic acid and its activity to destabilize Alzheimer's beta-amyloid fibrils in vitro

Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the destabilization of preformed fAbeta in the CNS would be attractive therapeutic targets for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) and wine-related polyphenols inhibit fAbeta formation from Abeta(1-40) and Abeta(1-42) as well as destabilizing preformed fAbeta(1-40) and fAbeta(1-42) dose-dependently in vitro. Using fluorescence spectroscopic analysis with thioflavin T and electron microscopic studies, we examined the effects of polymeric polyphenol, tannic acid (TA) on the formation, extension, and destabilization of fAbeta(1-40) and fAbeta(1-42) at pH 7.5 at 37 degrees C in vitro. We next compared the anti-amyloidogenic activities of TA with myricetin, rifampicin, tetracycline, and NDGA. TA dose-dependently inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42), as well as their extension. Moreover, it dose-dependently destabilized preformed fAbetas. The effective concentrations (EC50) of TA for the formation, extension and destabilization of fAbetas were in the order of 0-0.1 microM. Although the mechanism by which TA inhibits fAbeta formation from Abeta as well as destabilizes preformed fAbeta in vitro is still unclear, it could be a key molecule for the development of therapeutics for AD.     

Thyroid hormone and gamma-aminobutyric acid (GABA) interactions in neuroendocrine systems

Thyroid hormones (THs) have critical roles in brain development and normal brain function in vertebrates. Clinical evidence suggests that some human nervous disorders involving GABA(gamma-aminobutyric acid)-ergic systems are related to thyroid dysfunction (i.e. hyperthyroidism or hypothyroidism). There is experimental evidence from in vivo and in vitro studies on rats and mice indicating that THs have effects on multiple components of the GABA system. These include effects on enzyme activities responsible for synthesis and degradation of GABA, levels of glutamate and GABA, GABA release and reuptake, and GABA(A) receptor expression and function. In developing brain, hypothyroidism generally decreases enzyme activities and GABA levels whereas in adult brain, hypothyroidism generally increases enzyme activities and GABA levels. Hyperthyroidism does not always have the opposite effect. In vitro studies on adult brain have shown that THs enhance GABA release and inhibit GABA-reuptake by rapid, extranuclear actions, suggesting that presence of THs in the synapse could prolong the action of GABA after release. There are conflicting results on effects of long term changes in TH levels on GABA reuptake. Increasing and decreasing circulating TH levels experimentally in vivo alter density of GABA(A) receptor-binding sites for GABA and benzodiazepines in brain, but results vary from study to study, which may reflect important regional differences in the brain. There is substantial evidence that THs also have an extranuclear effect to inhibit GABA-stimulated Cl(-) currents by a non-competitive mechanism in vitro. The thyroid gland exhibits GABA transport mechanisms as well as enzyme activities for GABA synthesis and degradation, all of which are sensitive to thyroidal state. In rats and humans, GABA inhibits thyroid stimulating hormone (TSH) release from the pituitary, possibly by action directly on the pituitary or on hypothalamic thyrotropin-releasing hormone neurons. In mice, GABA inhibits TSH-stimulated TH release from the thyroid gland. Taken together, these studies provide strong support for the hypothesis that there is reciprocal regulation of the thyroid and GABA systems in vertebrates.     

Simultaneous ultrastructural demonstration of heavy metals (silver, mercury) and acid phosphatase

A histochemical technique which permits the simultaneous visualization of heavy metals and acid phosphatase at the ultrastructural level is described. The technique was applied to the anterior pituitary gland, the spinal cord and the liver. In all of the tested organs, both mercury and silver were found to accumulate primarily in the lysosomes, although small amounts of both metals could be observed in other organelles including endocytotic vesicles. In the anterior pituitary, few mercury deposits were found in the secretory granules.