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1.
Summary A model system of polyacrylamide films containing the Triton extract of rat brain homogenate was applied to investigate quantitatively some aspects of three methods for the cytochemical demonstration of acetylcholinesterase activity (Lewis 1961; Karnovsky and Roots 1964; Tsuji 1974).Biochemical determinations showed that about 90% of the acetylcholinesterase activity originally present in the Triton extract were still detectable in the films. The relationship of the formation of cuprous thiocholine iodide in the case of the methods of Lewis (1961) or Tsuji (1974) and of cupric ferrocyanide at the reaction of Karnovsky and Roots (1964) to either enzyme concentration or incubation time were tested in detail. The results showed that for the method of Tsuji and, with some restrictions, also for the method of Karnovsky and Roots a linearity exists in these two respects. In the case of the Lewis technique, an approximate linearity between the amount of reaction product and incubation time could only be found from 90 min onward, but no linearity was detected in relation to the enzyme concentration. At low enzyme concentrations, too little white precipitate was formed in comparison to higher ones. Therefore it is suggested that this technique, as compared to the methods of Tsuji and Karnovsky and Roots, probably is less suitable as a quantitative cytochemical method.This word was performed while one of us (Dr. Andrä) was in receipt of a visitor grant from the Netherlands Organization for the Advancement of Pure Research (ZWO) 相似文献
2.
Seung U. Kim 《Histochemistry and cell biology》1976,48(3):205-217
Summary Explants of 10–12 day chick embryo spinal cord were cultured by coverslip-roller tube method for 3–80 days. The cellular and subcellular localization of acetylcholinesterase activity in cultured neurons was studied by the thiocholine techniques of Karnovsky and Roots and Lewis and Shute.At the light microscopic level, acetylcholinesterase was demonstrated in the neurons of both ventral and dorsal horn regions. Occasionally neurons migrated in the outgrowth zone exhibited strong intracellular activity.At the electron microscopic level, acetylcholinesterase activity was found in the nuclear envelope, granular endoplasmic reticulum and the Golgi apparatus of the neurons. No enzyme reaction was detected in the glial cell cytoplasm. 相似文献
3.
P Kugler 《Histochemistry》1987,86(5):531-532
The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures. 相似文献
4.
Acetylcholinesterase, an enzyme responsible for hydrolyzing of acetylcholine to choline and acetic acid residues, is detected in the guard cell protoplasts. Extensive acetylcholinesterase activity has been found in the guard cell protoplasts as compared with the mesophyll cell protoplasts. Moreover, light could stimulate the enzyme activity. Localization of acetylcholinesterase in the stomata of Vicia faba L. was undertaken using Karnovsky and Roots cytochemical method. It was found that in the stomata of this plant products of acetylcholinesterase enzymatic reaction mainly appeared in the outer side of the guard cell ventral wall and inner wall. When the staining time was prolonged, products of acetylcholinesterase enzymatic reaction could also be found in the ventral and inner wall of the guard cells. In addition, more extensive product of enzymatic reaction was observed in the opened stomata than in the closed stomata. It was assumed that acetylcholineaterase may participate in the regulation of stomatal movement by hydrolyzing acetylcholine around the stomata. 相似文献
5.
P. Kugler 《Histochemistry and cell biology》1987,86(5):531-532
Summary The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain, a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1) 相似文献
6.
Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase 总被引:2,自引:1,他引:1
《The Journal of cell biology》1985,101(5):1930-1940
We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219- 221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE. 相似文献
7.
T M Holt 《The American journal of anatomy》1975,144(2):169-195
Some question exists concerning the ability of the embryonic tongue to undergo reflex movements at the time of palatal closure (15.5 days of development). Functional motor endplates are prerequisite for such movements to occur. Light and ultrastructural cytochemical methods were employed to elucidate the morphology of neuromuscular relationships in the developing mouse tongue. The A/Jax mice used in the experiments demonstrated a 12-20% incidence (seasonal variation) of spontaneous cleft palate, allowing a correlation between normal and teratological processes. Organized myofibrils were first seen in tongues of normal and spontaneous cleft lip-cleft palate (SCL-CP) specimens at 14.5 days of development. The thiocholine technique of Karnovsky and Roots was used to demonstrate acetylcholinesterase (AChE) activity at the light microscope level. The Lewis and Schute method was used for ultrastructural localization of this enzyme. Tissues from normal and SCL-CP specimens from 12.5 to 20.5 days of gestation failed to show differences in amounts or distribution of AChE activity. AChE activity was seen as early as 14 day's gestation. Electron microscopic studies demonstrated reaction product in the endoplasmic reticulum and nuclear envelope of developing myoblasts. AChE activity at the developing neuromuscular junction and the occurrence of myofilaments preceded palatal closure by several days. Based on these morphological and histochemical findings the tongue of normal and SCL-CP embryos appears capable of responding to a neurogenic stimulus at the time of palatal closure. The findings suggest that the tongue of animals exhibiting a spontaneous cleft palate is not actively involved in the etiology of this condition. 相似文献
8.
Acetylcholinesterase has been demonstrated in the spiral ganglion cells by the method of Karnovsky and Roots. After incubation for 24 h the content of AchE was measured with the help of a three-stage scale. 22% of the cells showed strong, 50% medium and 28% weak AchE activity. Within the spiral ganglion perikarya, the enzyme was equally distributed. 相似文献
9.
A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes 总被引:1,自引:0,他引:1
The "direct coloring" thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 micron. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the detection of multiple forms of AChE in polyacrylamide or agarose gels. 相似文献
10.
Summary The cytochemical determination of the activity of glucose-6-phosphate dehydrogenase (G6PDH) with tetranitro blue tetrazolium (TNBT) was studied with model films of polyacrylamide gel incorporating purified enzyme. This model system enabled a quantitative study to be made of different parameters involved with the cytochemical assay as it is applied to sections or smears. The enzyme activity of G6PDH incorporated in the model films was also assayed biochemically. Optimal conditions for retaining the maximum amount of enzymic activity are described. The behaviour of G6PDH towards enzyme inhibitors was found to be similar in model films and in solution. With TNBT, absorbance measurements at a single wavelength (535 nm) were used to estimate the enzyme activity quantitatively. When carried out under standardized conditions, both the cytochemical and biochemical assay showed a linear relation with the time of incubation and obeyed the Beer-Lambert law. The correlation between biochemical and cytochemical data was very high, which enabled cytochemical data to be converted into absolute units of enzyme activity. The data obtained in this way closely resembled the data of enzyme activity calculated from the absorbance of formazan produced inside polyacrylamide model films and afterwards extracted into a suitable solvent. 相似文献
11.
乙酰胆碱酯酶在蚕豆保卫细胞中集中分布 总被引:3,自引:0,他引:3
动物细胞中,乙酰胆碱酯酶负责把乙酰胆碱水解为胆碱和乙酸以起到终止信号的作用。蚕豆(ViciafabaL.)保卫细胞原生质体表面具有特异的水解乙酰胆碱的酯酶,光可以激活该酶的活性。组织学定位的结果显示,酶反应产物主要分布于气孔保卫细胞内壁和腹壁的外侧及内壁和腹壁中,表明一种类似于动物突触传递的机制可能存在于气孔保卫细胞和周围细胞之间,即乙酰胆碱发挥作用后由乙酰胆碱酯酶分解以终止其作用;此外开放的气孔周围具有更高的酶活性,说明乙酰胆碱酯酶可通过水解气孔周围的乙酰胆碱而调控气孔运动 相似文献
12.
A simple and reproducible method was used for the cytophotometric assay of alkaline phosphatase activity by end point measurements after incubation at 70 degrees C. Alkaline phosphatase was incorporated in polyacrylamide gel model films and its activity was demonstrated with a simultaneous coupling method. The initial reaction rate was 4.7 times faster than at 37 degrees C. At 37 degrees C, linear reaction rates were obtained up to 90 min incubation. Deviation from linearity occurred only when the amount of final reaction product precipitated inside the films was too high to be measured cytophotometrically. In that case, levelling off of the reaction rate was due to the out-of-range error of the cytophotometer. At 70 degrees C, reaction rates were distinctly non-linear from the onset of incubation. This was due to heat inactivation of the enzyme molecules. A plateau level was reached after approximately 60 min incubation, irrespective of the amount of enzyme incorporated, indicating that all enzyme molecules had become inactivated after this incubation period. The inactivation process followed first-order kinetics. The plateau value as well as the slope of the initial reaction were found to be linearly related to the amount of enzyme incorporated. Therefore, plateau absorbance values can be used as a relative measure of enzyme activity instead of initial reaction rates. This type of measurement could be valuable for routine applications of enzyme cytochemistry in diagnostic pathology, or when cytochemical reaction products are used as markers in immunocytochemistry or hybridocytochemistry. Precise control of incubation time is not necessary once the plateau value has been reached and preparations can be mounted and measured later. 相似文献
13.
N V Vladeeva 《Arkhiv anatomii, gistologii i émbriologii》1978,74(3):88-90
Karnovsky and Roots offer to use potassium ferricianide for coloured detecting the products of acetylcholine hydrolysis by cholinesterase. The method is based on the reduction of ferricianide to ferrocianide which forms with copper ions, present in the solution, unsoluble ferrocianide. Some properties of ferricianide ion, however, (stability, large size and great hydratation) make it difficult for the substance to penetrate the native cell membranes. The method by Karnovsky and Roots applied to laminated muscular tissue and to the rat nonfixed whole diaphragm, and to the sections from nonfixed tissue of the cat skeletal muscle verifies space isolation of ferricianide from the enzyme localized at the other side of cell membrane. 相似文献
14.
A histochemical method for the demonstration of acetylcholinesterase activity using semipermeable membranes 总被引:1,自引:0,他引:1
Summary The direct coloring thiocholine method of Karnovsky and Roots (1964) for the demonstration of acetylcholinesterase (AChE) activity was modified and adapted to the technique of semipermeable membranes. In this way it is possible to demonstrate histochemically both the bound as well as the soluble part of AChE activity. The localization of the reaction product is very distinct. Microdensitometric investigations of results of this method showed a linear increase of the amount of reaction product up to an incubation time of 180 min and section thickness up to 24 m. The medium supplemented with buffer (instead of agar) can be used for the demonstration of AChE activity in cryostat sections adherent to slides and is also very suitable for the defection of multiple forms of AChE in polyacrylamide or agarose gels.In honour of Prof. P. van Duijn 相似文献
15.
A K Raap 《Histochemistry》1986,84(4-6):317-321
Fluorescence enzyme cytochemical procedures will contribute significantly to biomedical problems where knowledge of the enzymic composition of individual cells is important. Compared with the number of absorbance enzyme cytochemical methods, relatively few fluorescence procedures have been reported. In this paper, the merits of the described methods are discussed. A distinction is made between methods with and without a capture reaction. Only a few methods satisfy the requirement of accurate localization of the final product and high signal to noise ratios. Thus, there still is a need for valid fluorescence cytochemical enzyme methods. It is concluded that the bottle neck for valid fluorescence cytochemical enzyme methods is the development of efficient fluorogenic capture reactions for the primary enzyme products. 相似文献
16.
A. K. Raap 《Histochemistry and cell biology》1986,84(4-6):317-321
Summary Fluorescence enzyme cytochemical procedures will contribute significantly to biomedical problems where knowledge of the enzymic composition of individual cells is important. Compared with the number of absorbance enzyme cytochemical methods, relatively few fluorescence procedures have been reported. In this paper, the merits of the described methods are discussed. A distinction is made between methods with and without a capture reaction. Only a few methods satisfy the requirement of accurate localization of the final product and high signal to noise ratios. Thus, there still is a need for valid fluorescence cytochemical enzyme methods. It is concluded that the bottle neck for valid fluorescence cytochemical enzyme methods is the development of efficient fluorogenic capture reactions for the primary enzyme products.In honour of Prof. P. van DuijnSupported (in part) by the Foundation for Medical Research (FUNGO), which is subsidized by the Netherlands Organization for the Advancement of Pure Research 相似文献
17.
Cholinergic innervation of the cornea and iris of the newborn and adult guinea pig was studied by the technique of Karnovsky and Roots (1964). The given structures are both richly innervated. The cholinesterase reaction of the cornea is more strongly positive in adult animals, whereas the intensity of the reaction of the iris in newborn and adult guinea pigs is almost identical. 相似文献
18.
A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cell is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used. 相似文献
19.
C. J. F. van Noorden J. Tas I. M. C. Vogels G. G. de Schepper 《Histochemistry and cell biology》1982,74(2):171-181
Summary A new method for enzyme cytochemical studies on individual cells is developed. Cells are incorporated in the matrix of a thin film of transparent polyacrylamide prior to incubation in a cytochemical medium. Five different kinds of individual cells, i.e. isolated rat hepatocytes, isolated mouse oocytes, cultivated human fibroblasts, rat thymocytes and human blood cells are used for testing the applicability of this method for the cytochemical demonstration of glucose-6-phosphate dehydrogenase with tetranitro BT. The incorporation technique solves at least some of the problems occurring with enzyme cytochemistry on single cells. The morphology of the cells is very well preserved, the formazan precipitation due to enzyme activity occurs entirely within the cell cytoplasm, the nothing dehydrogenase activity can be kept very low and the loss of cells is completely prevented with all cell types used. 相似文献
20.
THE LOCALIZATION OF ACETYLCHOLINESTERASE AT THE AUTONOMIC NEUROMUSCULAR JUNCTION 总被引:2,自引:1,他引:1
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Acetylcholinesterase has been localized at the autonomic neuromuscular junction in the bladder of the toad (Bufo marinus) by the Karnovsky method. High levels of enzyme activity have been demonstrated in association with the membranes of cholinergic axons and the adjacent membranes of the accompanying Schwann cells. The synaptic vesicles stained in occasional cholinergic axons. After longer incubation times, the membrane of smooth muscle cells close to cholinergic axons also stained. Axons with only moderate acetylcholinesterase activity or with no activity at all were seen in the same bundles as cholinergic axons, but identification of the transmitter in these axons was not possible. 相似文献