Demonstration Of Succinic Dehydrogenase Activity Research Articles

金属表面改質材アドロイ‐ＯＨ（Ｇａ‐Ｓｎ合金）の細胞毒性 第２報 純金円板表面に応用したアドロイ‐ＯＨの細胞毒性

We reported previously that the main dissolved element of Adlloy-OH in saline was gallium and had a slightly cytotoxic effect. In the present paper a cytotoxic effect of Adlloy-OH which was applied to a metal surface was investigated by the millipore filter method (Wennberg, A., 1979).Adlloy-OH which is liquid at room temperature was applied to the surface of a pure gold disk (7mm in diameter) with a pure tin bar. Surplus liquid alloy on the metal surface was wiped off with clean paper tissue. Metal disks to which Adlloy-OH was applied were used to the cytotoxicity test.A cell monolayer was established on a millipore filter which was placed on agar medium, cell side down. Test specimens were placed on top of millipore filter. The culture dish wasincubated for 2 hours at 37°C in a humidified atmosphere of 5 per cent CO2 in air. The cell reaction was assessed by incubating the cells, still adherent to the filter, for the demonstration of succinate dehydrogenase activity. The filters were examined macroscopically and scores from 0 to 3 were given to grade the severity of the cell responce.Adlloy-OH which was applied to the metal surface showed no toxic effect on the cells as a result.

Quantitative succinate dehydrogenase histochemistry in the hippocampus of aged rats

Quantitative histochemical methods (microphotometric kinetic and end-point measurements, and morphometric analyses of reactive areas) were used to investigate the levels of succinate dehydrogenase (SDH) in the hippocampus of young adult (3-6 months old) and aged male rats (24-27 months old). Methodological studies concerning the demonstration of SDH activity, which were performed using hippocampi of young animals, revealed a linear relationship between the reaction time and the amount of reaction product for up to 20 min; kinetic (continuous) and end-point measurements provided the same results. In a number of experiments, it was established that an incubation medium consisting of 100 mM succinate, 10 mM sodium azide, 3 mM nitro blue tetrazolium chloride, 0.25 mM phenazine methosulfate, and 7.5% polyvinylalcohol in 0.05 M Hepes buffer (final pH 7.5) was optimal for quantitative SDH histochemistry in the hippocampus. Comparative quantitative investigations of SDH activity in rat hippocampi showed that, in most regions and layers of the hippocampus of both young and aged rats, the levels of SDH activity increased along the rostrocaudal axis of the hippocampus, i.e., higher levels were present in the caudal than in the rostral pole. In both groups, the highest SDH levels were observed in the molecular layer of the cornu ammonis (CA)-1, the CA-3, and the fascia dentata (middle and outer thirds), most of which are termination fields of the excitatory perforant path arising from the regio ento-rhinalis. Furthermore, in almost all of the investigated layers, the older animals exhibited lower SDH levels than young animals. These differences were statistically significant in the molecular layer of the fascia dentata and in most layers of the CA-3. The lower SDH levels in aged animals are discussed in relation to the reduced capacity for energy metabolism in the aging brain.

A method for toxicity screening of biomaterials using cells cultured on millipore filters

In the present paper a tissue culture technique is described whereby the toxicity of setting and solid materials may be evaluated. A cell monolayer was established on a millipore filter which was placed on an agar medium, cell side down. Test specimens were placed on top of the millipore filter and were allowed to influence the cells through the filter for two hours. The cell reaction was assessed by incubating the cells, still adherent to the filter, for the demonstration of succinate dehydrogenase activity. Materials with a cytotoxic effect caused a zone of inhibited enzyme activity in the cell-material contact area. The filters were examined macroscopically and scores from 0 to 3 were given to grade the severity of the cell response. Unset and set silicate cement, zinc phosphate cement and an acrylic resin were tested. The results obtained were consistent and in accordance with those of previous reports. The method was simple and rapid and appeared suitable for the assay of larger test series.

An x-ray microanalytical study of the ferricyanide reaction for the electron cytochemical demonstration of succinate dehydrogenase activity in isolated mitochondria.

The cytochemical technique for the demonstration of succinate dehydrogenase activity by ferricyanide reduction and simultaneous coupling with Cu2+ has been investigated by the combined techniques of transmission electron microscopy and X-ray microanalysis. The localization of the final reaction product of succinate dehydrogenase activity within isolated mouse liver mitochondria is described, and variations in the composition of the final reaction product are shown to occur, both at various sites within the mitochondrion, as well as after different reaction times.

Glycol methacrylate embedding in enzyme histochemistry: application to arthropode tissue incubated for demonstration of unspecific esterase and succinic dehydrogenase activity.

The final products of unspecific esterase and succinic dehydrogenase were demonstrated in 1–2 μm sections of tick salivary glands embedded in glycol methacrylate (GMA). In the esterase experiments, the tissue specimens were incubated after fixation in glutaraldehyde or acrolein, and then embedded in GMA. For demonstration of succinic dehydrogenase activity, the specimens were incubated prior to glutaraldehyde fixation followed by embedding in GMA. In sections of all preparations intense enzymatic reaction was observed. High resolution light microscopy could efficiently be used for precise locating of the enzymic products, due to excellent morphologic reference in semithin GMA sections.

Histochemical demonstration of succinic dehydrogenase activity during sequential molar development in the Swiss albino mouse.

Applying the ditetrazolium salt (Nitro-BT) method for succinic dehydrogenase, murine molar teeth were studied sequentially from the cap stage of development through the appositional stages of odontogenesis. Reaction-distribution and intensity varied relative to the developmental stage as well as the zone of maturation within a given stage. The peripheral cells of the parent dental lamina exhibited some activity, as did the outer enamel epithelium of the bell stage. During the period of matrix apposition, components of the stratum intermedium, ameloblastic zone and odontoblastic layer region of the dental papilla demonstrated intense enzymatic activity. Cells actively engaged in enamel matrix production demonstrated activity in the basal and distal cell segments. High activity continued in the papillary layer of the enamel organ, as well as in the cells of the dental sac during the postamelogenic period.

The effect of dimethyl sulfoxide (DMSO) in the incubation medium for the cytochemical localization of succinate dehydrogenase

Addition of dimethyl sulfoxide (DMSO) to the cytochemical incubation medium for succinate dehydrogenase was attempted to accelerate penetration with consequent shortening of the incubation time. The copper-ferrocyanide medium for demonstration of succinate dehydrogenase activity was applied to fresh and hydroxyadipaldehyde-fixed muscle of the hamster and mouse and the albumen secreting gland cells of the hen oviduct. Cytochemical evidence indicated that DMSO did not seem to inhibit this enzymatic activity. With a shorter incubation time, less heterogeneity in reaction product was obtained in the mitochondria of muscle. The marked heterogeneity found in the reaction in the intracristal space of mitochondria also was diminished with addition of DMSO to the medium. The gland cells, whose ultrastructure was not well preserved with prolonged incubation, showed reductase reaction with the DMSO-containing medium.

Electron-microscopic-histochemical demonstration of succinic-dehydrogenase activity in root cells of yellow lupine

Succinic dehydrogenase (SDH) activity was demonstrated in unfixed root segments from Lupinus luteus at the ultrastructural level by the use of ferricyanide as electron acceptor. The specificity of the reaction was proven by malonate inhibition. The reaction product was found to be localized in the mitochondria and to a lesser extent on the membranes of plastids. Different mitochondria of the same cell often showed different intensity of the staining reaction. Different cells of the same tissue exhibited varying degrees of enzyme activity. An increase was found in the number of cells exhibiting the SDH reaction, as well as in the intensity of the reaction itself, from the meristematic zone of the root to the more differentiated regions.

The use of ferricyanide for the light and electron microscopic demonstration of succinic dehydrogenase activity.

The conditions of the use of ferricyanide in the histochemical demonstration of succinic dehydrogenase activity (SDH) were investigated. The method is based on a simultaneous coupling reaction in which the ferrocyanide produced is captured by copper at the site of enzyme activity. The choice of an appropriate chelator to prevent the precipitation of copper by other components of the incubation medium was shown to be crucial. In the present study tartrate was found to give good results. The localization obtained is of high accuracy, no nonspecific attachment of the chemicals to tissue components could be detected. The highly electron dense final reaction product was found to cover the mitochondrial membranes as distinct granules of 40–80 A diameter separated by 30 A intervals. The side by side occurrence of mitochondria exhibiting normal, less, and no activity was confirmed in various tissues. The precipitate may be identified also in the light microscope owing to its dull brown colour, but for routine light microscopic purposes the precipitation of silver on its surface is recommended.

The use of tetrazolium salts in the histochemical demonstration of succinic dehydrogenase activity in plant tissues.

Succinic dehydrogenase activity was determined in fresh or cryostat sections of tissues from Allium cepa, Vicia faba, Pisum sativum and Helianthus tuberosus using different tetrazolium salts as electron accepters. In 10 μ fresh or frozen sections a reaction was obtained with TNBT, NBT, MTT, and INT but not with NT, BT or TTC. In contrast a reaction was obtained with each of the tetrazolium salts in 50–150 μ fresh or frozen sections. The observed differences in the abilities of the tetrazolium salts to demonstrate succinic dehydrogenase activity are discussed.

Ubiquinone and phospholipids as limiting factors in the histochemical demonstration of succinic dehydrogenase activity.

Deposition of ubiquinone and preferably of a crude phospholipid mixture and ubiquinone on cryostat sections of mouse kidney, liver and heart increased the intensity of staining for succinic dehydrogenase. A similar treatment partly restored the activity of this enzyme which was reduced by exposure to 60°C, long storage at 37°C, oxidation and treatment with lipid solvents. The restoration was present only in the case of mild noxious treatments. It is suggested that removal or structural alteration of ubiquinone and phospholipids may limit the rate of dehydrogenase activity; more drastic treatments affect the enzyme itself so that these factors cannot exert any restorative activity.

Histochemical Demonstration of Succinic Dehydrogenase Activity of the Hair Cells by Means of Intravital Perfusion of the Cochlea

Intravital incubation (for 15 minutes) of the perilymphatic space of the cochlea with substrate (nitro blue tetrazolium) histochemically could demonstrate succinic dehydrogenase activity of the hair cells of the organ of Corti in living guinea pigs. Subsequent surface preparation of the organ of Corti provided good surface specimens for observation of succinic dehydrogenase activity of the hair cells. Twenty guinea pigs of the Hartley strain were used. Fifteen of them were exposed for 8 hours daily to a sound of 4, 000c.p.s. of an intensity of 80dB for 8 days. The other 5 were the control. The threshold of the pinna reflex of the animals was estimated by the audiometer for animals before and after the exposure. One of the experimental animals exposed to the sound showed slight hearing impairment. Histochemically there were no distinct differences in succinic dehydrogenase activity of the hair cells between in the control and experimental animals. Both the inner and outer hair cells contained a large amount of diformazan granules in the supra-, infra- and perinuclear areas of the cytoplasms. There was no remarkable difference in succinic dehydrogenase activity between the upper and lower turns.

The histochemical demonstration of succinic dehydrogenase activity in developing teeth.

The histochemical demonstration of succinic dehydrogenase activity in developing teeth.

Histochemical demonstration of succinic dehydrogenase activity in osteoclasts.

A REVIEW of the literature has revealed but few studies on enzyme characteristics of osteoclasts. None of these, however, related to dehydrogenase activity.

Improved technique for the histochemical demonstration of succinic dehydrogenase in brain tissue.

A tissue cutting technique for the histochemical demonstration of succinic dehydrogenase activity in brain tissue is described. The demonstration of succinic dehydrogenase in fine and delicate structures depends on a tissue temperature of –3 to –2°C. during cutting. Artefacts such as coarse granules, loss of activity and diffusion of stain are prevented by minimizing of traumatic effects due to this improved technique.