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PubMed1980

Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles.

A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate D-fructose-6-phosphate to D-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and D-glyceral-dehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-D-glycerate. Concomitantly the electrons are transported via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.

Anaerobiosis↗
PubMed1979

A quantitative cytochemical study of glucose-6-phosphate dehydrogenase and delta 5-3 beta-hydroxysteroid dehydrogenase activity in the membrana granulosa of the ovulable type of follicle of the rat.

During the last four days of follicular development prior to ovulation, the activities of delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta OHD) and glucose-6-phosphate dehydrogenase (G-6-PD) were quantified in cryostat sections of the rat ovary. The product of the enzyme reactions were measured using a scanning and integrating microdensitometer. The enzyme activity was measured in the peripheral region, the antral region and the cumulus of the membrana granulosa (MG) of these follicles on the morning of each of the four days of the estrous cycle. G-6-PD activity was measured in the presence and absence of an intermediate hydrogen acceptor, phenazine methosulphate, to provide a measure of the quantity of Type I and Type II Hydrogen (H) generated: Type I H is considered to be related to hydroxylating reactions such as those of steroids and Type II H to other general biosynthetic activities of cells. In all three regions of the MG of follicles of the ovulable type, 3 beta OHD activity was lowest in estrus and diestrus-1, increased on diestrus-2 and peaked in proestrus. In estrus and diestrus-1, the level of 3 beta OHD activity in the three regions was comparable. However, by diestrus-2, and even more conspicuously in proestrus, enzyme activity was significantly greater in the peripheral region than in the antral region or in the cumulus. During the same period, the level of enzyme activity remained comparable in the last two regions. Throughout the estrous cycle, both Type I and Type II H generation from G-6-PD was greatest in the peripheral region, less in the antral region and least in the cumulus. In the eripheral region, Type I H generation increased progressively after diestrus-1, to reach a maximum in prestrus. In the antral region, Type I H generation increased between diestrus-1 and diestrus-2 and then remained unchanged through proestrus. In the cumulus, Type I H generation remained at levels seen in estrus throughout the remainder of the cycle. Generation of Type II H, in the peripheral region was constant throughout the estrous cycle. In contrast, in the antral region and cumulus, Type II H generation was greater in diestrus-1 and diestrus-2 than on either proestrus or estrus.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

The histochemistry of developing adipocytes in primary stromal-vascular cultures of rat adipose tissue.

Histochemical analysis for NADP-dependent dehydrogenases, succinate dehydrogenase, NADH and NADPH- tetrazoleum reductases and esterase was conducted on primary cultures of adipose tissue stromal-vascular cells. Enzyme activities were restricted to clusters of lipid laden cells (adipocytes). The number of enzyme reactive adipocytes increased with length of culture. Coverslips were partially coated with collagen to allow comparisons of cell differentiation on coated (C-glass) and uncoated glass (U-glass) surface. There were no reactions for NADH- and NADPH- tetrazoleum reductases (TR) in cells on C-glass whereas adipocytes and stromal cells on U-glass were reactive. Glucose-6-phosphate (G6PDH) and 6-phosphogluconate (6PGDH) dehydrogenase activities were markedly demonstrated in both stromal cells and adipocytes on U-glass. Malate (MDH) and isocitrate (ICDH) dehydrogenase activities were higher in adipocytes than in stromal cells on the U-glass. Stromal cells on C-glass were either devoid of these enzymes (G6PDH, MDH, 6PGDH, ICDH) or activity was restricted to a small area of the cytoplasm. There were two levels of staining intensity in (MDH, ICDH, G6PDH, 6PGDH) adipocyte clusters on C-glass. Elimination of phenazine methosulphate from the NADP-dependent dehydrogenase medias and SDH media, caused a reduction in enzyme reactive adipocytes on the C-glass. This manipulation did not reduce the number of enzyme reactive cells on U-glass. Cells on C-glass and U-glass were distinctly different in esterase stained coverslips. These studies demonstrated enzyme histochemical reactions of adipocytes and stromal cells in primary culture that were dependent on the type of extracellular matrix. Furthermore, enzyme histochemistry was shown to be useful for delineating adipocytes from stromal cells in primary cultures.

Adipose Tissue↗
PubMed1975

Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. V. Isocitrate: NADP+ oxidoreductase (decarboxylating) and malate: NADP+ oxidoreductase (decarboxylating).

Improved histochemical techniques for the demonstration of NADP+-specific isocitrate dehydrogenase and malate dehydrogenase in tissue sections are described. With these techniques a semipermeable membrane is interposed between the incubating solutions and the tissue sections preventing diffusion of enzymes into the medium during incubation. In the histochemical system the NADP+-dependent enzymes catalyze the electron transfer from threo-Ds-isocitrate or L-malate into NADP+. Phenazine methosulphate and menadione serve as intermediate electron acceptors between reduced coenzyme and nitro-BT. Sodium-azide and amytal are incorporated into the incubating-medium to block electron transfer to the cytochromes. For demonstrating enzyme activities in sections containing non-specific alkaline phosphatase, a phosphatase inhibitor is added into the incubation media. Problems involved in the histochemical demonstration of both enzymes are discussed.

Alkaline Phosphatase↗
PubMed1988

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.

Aging↗
PubMed1988

Histochemical technique for the demonstration of pyruvate kinase activity.

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.

Adenosine Diphosphate↗
PubMed1985

Depletion of secondary lysosomes in mouse macrophages infected with Leishmania mexicana amazonensis: a cytochemical study.

Leishmania amastigotes lodge and multiply within parasitophorous vacuoles, which can fuse with secondary lysosomes of the host macrophages. This study examines the effect of infection with amastigotes of L. mexicana amazonensis on the secondary lysosomes of mouse macrophage cultures. The cultures were stained for the activities of two lysosomal enzyme markers, acid phosphatase and arylsulfatase, and the light microscopic observations were supplemented by electron microscopy. Nearly all noninfected macrophages contained numerous stained secondary lysosomes. The number of such lysosomes was markedly reduced 24 h postinfection, and the reduction persisted for at least 10 days. Stained secondary lysosomes reappeared after the amastigotes were destroyed by exposure of the cultures to phenazine methosulfate or by placing them at 37.5 degrees C. The depletion of lysosomes shown by cytochemical methods may reflect a high rate of fusion of the lysosomes with the parasitophorous vacuoles, exceeding the rate of formation of new secondary lysosomes. Alternatively, the parasites may inhibit the synthesis of lysosomal hydrolases, or the assembly or formation of primary or secondary lysosomes.

Acid Phosphatase↗
PubMed1985

The histochemical demonstration of fructose diphosphate aldolase activity using a semipermeable membrane technique.

In this communication an enzyme histochemical multistep technique for the demonstration of class 1 fructose-1,6-diphosphate aldolase in heart and skeletal muscle sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the enzyme into the medium during incubation. In the histochemical system the enzyme cleaves the substrate D-fructose-1,6-diphosphate to dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triose phosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase and the electrons are transported concomitantly via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.

Animals↗
PubMed1988

On the role of oxygen in dehydrogenase reactions using tetrazolium salts.

Fundamental aspects of the reduction of tetrazolium salts were investigated and, in particular, the role of oxygen in the reduction. It was found that oxygen had a competitive inhibitory effect on the reduction of (Tetra)Nitro BT mediated by NADH and phenazine methosulphate. This competitive effect, under aerobic conditions, could be reversed by using tetrazolium concentrations of 5 mM. Oxygen did not have a significant effect on BPST reduction, whereas the inhibitory effect of oxygen on the reduction of Neotetrazolium was not reversed by increasing the tetrazolium concentration. The oxygen effect on Nitro BT reduction was considerably less when macromolecular substances such as albumin or polyvinyl alcohol were added to the medium. This may be due to increased Nitro BT concentrations being built up at the surface of macromolecules due to the nonpolar components of the Nitro BT molecule. When demonstrating glucose-6-phosphate dehydrogenase activity in vitro or in tissue sections with the use of Nitro BT, oxygen also had a direct inhibitory effect, even when azide was added to the medium for the inhibition of flavoprotein-mediated electron transfer to oxygen. Again, this direct inhibition of Nitro BT reduction by oxygen could be excluded by using a high Nitro BT concentration. Macromolecules present in the incubation medium or in tissue sections counteracted the oxygen effect. It is concluded that the maximum reaction rate and optimum localization of dehydrogenases is obtained when histochemical media are used containing 5 mM (Tetra)Nitro BT and 20% polyvinyl alcohol.

Animals↗
PubMed1978

A quantitative histochemical study of three oxidative enzymes in solar keratoses and Bowen's disease.

Succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH) activities have been studied using quantitative enzyme histochemical techniques in the epidermis of five patients with solar keratoses and Bowen's disease. 'Non sun-exposed' buttock skin was compared with the skin from the actual lesion and adjacent, clinically normal paralesional skin. SDH activity was significantly increased in the basal layer and decreased in the granular layer in the epidermis of both lesion and paralesional skin, although the total epidermal activities were unchanged when compared to 'non-exposed' buttock skin. G6PDH activity was increased in the granular layer of paralesional epidermis and of lesions. No change in LDH activity was detected. Inclusion of phenazine methosulphate in the reaction mixtures resulted in a three-fold increase in formazan deposition without altering the localization. It is concluded that the quantitative changes and alteration in localization of SDH and G6PDH reported in solar keratoses are accompanied by similar changes in adjacent, clinically normal 'sun-exposed' skin and differ from normal 'non-exposed' skin. It is suggested that these changes may precede the development of the solar keratoses and that these findings may indicate a significant metabolic alteration in the events that lead to neoplasia.

Aged↗
PubMed1984

Kinetic behaviour of succinate dehydrogenase of three fibre types in skeletal muscle. I. Effects of temperature and a competitive inhibitor.

The kinetic behaviour of succinate dehydrogenase [EC 1.3.99.1] in three fibre types of rat gastrocnemius was examined by a quantitative histochemical method without disruption of the cellular structure. 2-(2-Benzothiazolyl)-3-(4-phthalhydrazidyl)-5-styryl-t etrazolium chloride (BPST) and phenazine methosulphate were used as electron acceptors. On measurement of the absorbance value at 530 nm of BPST formazan, produced by the succinate dehydrogenase reaction in sections, it was found that the staining intensity of succinate dehydrogenase was linearly proportional to both the incubation time and the thickness of the slice therefore, the initial velocity of the staining could be calculated. By Michaelis-Menten (1913) treatment of the dependence of the initial velocity on the substrate concentration in the absence and the presence of a competitive inhibitor, malonate, the Km and Vmax values for succinate and the Ki value for malonate were obtained. The Km and Ki values of the three fibre types were similar. The ration of the Vmax values of type A, B and C fibres was 1.0:2.0:3.3. The temperature dependence of the kinetic parameters was very similar in the three fibre types. These findings confirm that the differences in the staining intensity of the three fibre types reflect differences in the amounts, but not the properties, of succinate dehydrogenase.

Animals↗
PubMed1975

A permeability test for the study of mitochondrial injury in in vitro cultured heart muscle and endothelioid cells.

A cytochemical permeability test for the detection of injury to in situ mitochondria of cultured heart cells is presented. The test is based on the increased rate at which injured mitochondria stain for succinate dehydrogenase activity. Whereas an intact inner mitochondrial membrane limits the rate at which Nitro Blue tetrazolium and phenazine methosulphate reach succinate dehydrogenase, injured mitochondria allow these reactants to reach the enzyme more rapidly to form microscopically-observable formazan granules. The extent of staining at fixed durations of incubation with the reactants was assessed on a blind basis with pseudo dark-field microscopy, using a standardized rating scale. Differences in the staining of control and treated cells were analysed statistically by a semi-quantitative method. Treatment of the cultures with either vitamin A or chlorpromazine, resulted in more rapid mitochondrial staining. Brief pre-fixation of the cells with cold acetone also labilized the mitochondria as did a delay in the change of culture medium.

Animals↗
PubMed1991

Glucose handling by hepatocytes from obese Zucker rats.

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-14C]glucose and [U-14C]lactate incorporation into [14C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of 3H2O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [14C]glycogen or 14CO2, the [14C] total uptake was significantly higher in the obese animals. The high rate of [14C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-14C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-14C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-14C]glucose/[U-14C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.

Animals↗
PubMed1988

Simultaneous histochemical assay of two dehydrogenases in the same cell.

A new approach has been developed for the simultaneous assay of the activities of two enzymes (lactate and succinate dehydrogenases) in the same cell in sections of unfixed liver. The sections, mounted on coverslips, were placed on top of 0.6-mm thick 0.8% low gelling-temperature agarose films containing the substrates of both enzymes (70 mM lactate and 50 mM succinate, respectively) plus 80 mM Tris-HCl buffer (pH 7.5), 5 mM EDTA, 10 mM NaN3, 1.5 mM NAD+, 1.2 mM Nitro BT and 0.26 mM phenazine methosulphate. The integrated absorbance (A) at 585 nm of the final reaction product formazans deposited by the two enzymes in a selected hepatocyte was measured continuously at 37 degrees C as a function of incubation time, using a Vickers M85 microdensitometer. The intercept A0 on the A-axis of the linear regression line of A on time was determined. After a known incubation time t, the absorbance A1, was noted and the section placed on another gel film lacking the substrates in order to estimate the final reaction product either formed in the gel film or lost from the cell. The absorbance A2 of the hepatocyte was remeasured. The reaction velocities (activities) vL and vS of lactate and succinate dehydrogenases, respectively, were calculated from the following equations: vL = [(A1-A2) - A0(1- alpha L)]/(1-alpha L)t and vS = (A2-alpha LA1)/(1-alpha L)t where alpha L = A2/A1 for hepatocytes incubated on gel films containing only lactate as the substrate. This parameter was found to be virtually constant (0.44) over a wide range of vL.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1999

Antioxidant-Angioprotective Actions of Calcium Dobesilate in Diabetic Rats.

Calcium dobesilate was tested in rats with streptozotocin(STZ)-induced diabetes, for its angioprotective properties against the extravasation of plasma Evans blue towards the peritoneal cavity, induced in situ by the free radical generating agent phenazine methosulfate (PMS). In diabetic rats pretreated with a single oral dose of 50 and 200 mg/kg calcium dobesilate, PMS-induced Evans blue extravasation was respectively reduced by 60 and 100% with respect to values in vehicle-treated animals (p < 0.05 and p < 0.01 respectively; in diabetic rats, PMS-induced Evans blue peritoneal extravasation was double as in control animals = 0.0814 +/- 0.019 vs. 0.0396 +/- 0.0083 h-1, p < 0.05). In diabetic rats pretreated with 50 and 100 mg/kg/day of calcium dobesilate for 7 days, PMS-induced Evans blue extravasation was respectively reduced by 56 and 80% with respect to values in vehicle-treated animals (p < 0.01 and p < 0.001 respectively). In conclusion, calcium dobesilate p.o. significantly and dose-dependently antagonized the increase in capillary permeability induced by an oxidative stress in the peritoneal cavity of diabetic rats. These results further suggest that the antioxidant properties of calcium dobesilate can be involved, at least in part, in its angioprotective actions in humans.

Journal Article↗
PubMed1987

[Significance of Pseudomonas aeruginosa virulence factors for acute and chronic Pseudomonas aeruginosa infections].

The virulence of Pseudomonas aeruginosa is multifactorial and caused by several extracellular enzymes and other substances. The importance of these virulence factors for the pathogenesis of human P. aeruginosa infections is dependent on the type of infection. For acute, systemic infections in immunocompromised patients, exotoxin A, alkaline protease and elastase are essential virulence factors. In localized infections (e. g., cystic fibrosis) they seem to be of minor importance, since they are neutralized by specific antibodies in immune complexes, and in the case of exotoxin A cleavage by proteinases from polymorphonuclear leukocytes occurs. The rhamnolipid of P. aeruginosa which has been detected in sputa of patients with cystic fibrosis evades the host's immune response and has to be regarded as a potential virulence factor together with the phenazine pigments, also in chronic P. aeruginosa infections.

ADP Ribose Transferases↗
PubMedDate not supplied

[Significance of Pseudomonas aeruginosa virulence factors in acute and chronic Pseudomonas aeruginosa infections].

The virulence of Pseudomonas aeruginosa is multifactorial and caused by several extracellular enzymes and other substances. The importance of these virulence factors for the pathogenesis of human P. aeruginosa infections is dependent on the type of infection. For acute, systemic infections in immunocompromised patients, exotoxin A, alkaline protease and elastase are essential virulence factors. In localized infections (e.g., cystic fibrosis) they seem to be of minor importance, since they are neutralized by specific antibodies in immune complexes, and in the case of exotoxin A cleavage by proteinases from polymorphonuclear leukocytes occurs. The rhamnolipid of P. aeruginosa which has been detected in sputa of patients with cystic fibrosis evades the host's immune response and has to be regarded as a potential virulence factor together with the phenazine pigments, also in chronic P. aeruginosa infections.

ADP Ribose Transferases↗
PubMed1987

A fluorescent redox dye. Influence of several substrates and electron carriers on the tetrazolium salt-formazan reaction of Ehrlich ascites tumour cells.

This study was performed to elaborate the best conditions for measuring the redox activity of Ehrlich ascites tumour cells by using a new tetrazolium salt, cyantolyl tetrazolium chloride (CTC). This tetrazolium salt forms a fluorescent water-insoluble formazan on reduction on the surface of intact vital cells. The influences of fixation and of various substrates and electron carriers on the cellular reduction of CTC were investigated quantitatively using an elution technique. The amount of formazan obtained after incubating vital cells with Meldola Blue as electron carrier was greater than that obtained with Methylene Blue, menadione, 2,6-dichloroindophenol, 1-methoxyphenazine methosulphate or phenazine methosulphate. Using flow cytometry, the formazan production per cell and, after staining the nuclear DNA, the distribution of the redox activity in the cell population can be visualized with satisfactory resolution. We conclude from our findings that dehydrogenases are only partially involved in the reduction of tetrazolium salts by intact cells and that a redox activity, probably related to a cell membrane-bound NAD(P)H-oxidase system, is mainly measured.

Animals↗