Biomedical subjects

F Thoné

Publications and source records attributed to F Thoné.

At least 19 recordsLinked to original sources

PubMed1992

Species differences in adenosine metabolic sites in the heart.

5'-Nucleotidase and purine nucleoside phosphorylase, two key enzymes in nucleoside metabolism, have been localized electronmicroscopically in left ventricular myocardium of the human, dog, pig, rabbit, guinea pig and rat. Ectonucleotidase activity was present in all species at the plasma membrane of pericytes. Reactive endothelial cells in the microcirculatory bed were restricted to those covering resistance arterioles. Cardiomyocytes were reactive only in the rat. Purine nucleoside phosphorylase was localized uniformly in the vascular endothelium of all species. The strongest activity was seen in the pericytes of guinea pig, rat and dog. Pericytes of rabbit and pig were virtually unreactive, whereas a minority of cells in human samples were positive. Cardiomyocytes were unreactive in all species. These variations in the distribution pattern of adenosine metabolic sites may have definite consequences for disposal and recovery of adenylates and their breakdown products in ischaemia and for the effects to be expected from interference with nucleoside transport inhibition.

5'-Nucleotidase↗

PubMed1992

Effects of R 56865 and phenytoin on mechanical, biochemical, and morphologic changes during ouabain intoxication in isolated perfused rabbit heart.

The Na+/Ca2+ overload inhibitor R 56865 (N-[1-[4-(4-fluorophenoxy)-butyl]-4-piperidinyl)-N-methyl-2- benzothiazolamine) has been reported to prevent or attenuate ischemia- as well as ouabain-induced cellular sodium and calcium load. We investigated the potency of this compound in preventing mechanical, biochemical, and ultrastructural consequences of ouabain (OUA) intoxication in isolated rabbit heart. The protective effect of the digitalis antidote phenytoin (PHT) on the consequences of ouabain intoxication was examined for comparison. In isolated perfused rabbit heart, OUA (0.4 microM) caused an increase in left ventricular end-diastolic pressure (LVEDP) that was accompanied by depletion of high-energy phosphates (80% less than in control), accumulation of tissue lactate (12-fold) and damage of contractile elements and mitochondria. Accumulation of lactate was associated with a decrease in oxygen consumption by the isolated perfused heart. R 56865 (1.0 microM) and phenytoin (60 microM) prevented increase in LVEDP, breakdown of the energy-rich phosphates creatine phosphate (CrP) and ATP, accumulation of lactate, and morphologic changes induced by OUA. The above-mentioned toxic effects of OUA are interpreted as consequences of mitochondrial failure finally leading to breakdown of the oxidative phosphorylation. Thus, we conclude that the protective action of both compounds, R56865 and PHT, may be attributed to prevention or attenuation of mitochondrial failure due to OUA-induced disturbance of ion homeostasis.

Adenosine Triphosphate↗

PubMed1991

Protective effects of R 56 865 against ischemic damage in the isolated rabbit heart.

We examined the role of calcium in the pathogenesis of ischemic cardiac cell death in the isolated working rabbit heart subjected to normothermic global ischemia followed by reperfusion. Apart from measurements of cardiodynamic function and ultrastructural examination, we also used a cytochemical procedure to localize exchangeable calcium pools at the ultrastructural level. The effects of verapamil (1.5 x 10(-8) M, 3 x 10(-8) M) (high affinity for L-type calcium channels) were compared with those of R 56 865 (4 x 10(-7) M) (Ca2+ overload blocker with low affinity for the L-type calcium channels). A severe depression of cardiac function was observed after solvent or verapamil pretreatment and 25 min of ischemia followed by reperfusion. R 56 865 treatment resulted in a significantly improved postischemic recovery when compared to solvent and verapamil treatment groups. The ultrastructural and cytochemical results corroborated the hemodynamic findings. In solvent and verapamil-treated hearts, irreversible damage was observed mainly in mid- and endocardial areas. Ultrastructural changes were accompanied by shifts in calcium localization: i.e. loss of sarcolemmal calcium binding capacity, accumulation of calcium precipitate in the mitochondria. In the R 56 865 treatment group, damage was limited to some cells scattered in the midcardial areas. In conclusion, R 56 865, which has little affinity for the slow channels was highly effective in protecting against ischemic damage, indicating that, in this experimental set-up, the calcium responsible for cellular Ca2+ overload is not entering via L-type calcium channels.

Animals↗

PubMed1991

Stunned myocardium has increased mitochondrial NADH oxidase and ATPase activities.

Ten anesthetized, open-chest dogs were subjected to occlusion of the left anterior descending coronary artery for 15 minutes, followed by reperfusion for 150 minutes. Hemodynamics were recorded and regional myocardial contraction was measured sonometrically. The hearts were then fixed in situ using glutaraldehyde for cytochemical studies. Systolic wall thickening remained unchanged in the non-ischemic myocardium, but was significantly depressed (stunned) in the area of the left anterior descending coronary artery during reperfusion. NADH oxidase and ATPase activities were very weakly present in mitochondria from non-ischemic myocardium. In the ischemic endocardium, irreversibly injured cells had mitochondria which were severely altered and contained no reaction products to the two enzymes. In contrast, high NADH oxidase and ATPase activities were present in mitochondria from the less severely injured cells of the endocardial zone of stunned areas. Since this zone is particularly susceptible to ischemia in dogs, the high mitochondrial NADH oxidase and ATPase activities may be early signs of ischemic damage, reflecting a disturbance in mitochondrial respiratory activity in stunned myocardium.

Adenosine Triphosphatases↗

PubMed1990

Cytochemical evidence of NADH-oxidase activity in the isolated working rabbit heart subjected to normothermic global ischaemia.

The cytochemical localization of NADH-oxidase, a possible source of oxygen derived toxic species was studied in the isolated working rabbit heart subjected to normothermic global ischaemia. The activity of this oxidase could be important for the damage observed during ischaemia, when cellular defence mechanisms against free radicals are depleted. In non-ischaemic myocardium only small amounts of the NADH-oxidase reaction product were present in the mitochondria. Although the reaction product could already be observed after 45 min of incubation, prolonged incubation times up to 2h were necessary to clearly define these reactive sites. The reaction product is substrate dependent and is not affected by cyanide. Exposure of the hearts to ischaemia resulted in an alteration of the enzyme activity depending on the degree of ischaemic damage. In ultrastructurally slightly altered areas a high degree of activity was observed in the mitochondria. In infarcted tissue, mitochondria contained little or no reaction product. This cytochemical study supports the hypothesis that hydrogen peroxide and oxygen radicals produced in the mitochondria by NADH-oxidase activity may contribute to the mitochondrial damage observed during ischaemia when NADH is no longer oxidized by the respiratory chain and cellular defence mechanisms are impaired.

Animals↗

PubMed1990

Singlet oxygen and myocardial injury: ultrastructural, cytochemical and electrocardiographic consequences of photoactivation of rose bengal.

Photoactivation of rose bengal leads to the generation of reactive oxygen intermediates (predominantly singlet oxygen with some superoxide anion) which are potentially injurious to biological systems. Isolated rat hearts were perfused aerobically at 37 degrees C with bicarbonate buffer for 10 min without rose bengal and for 10 min with rose bengal (500 nM). During the last 5 min of perfusion with rose bengal, hearts were globally illuminated (5500 lux) with light (530 to 590 nm) and electrocardiographic changes were detected within 2.7 +/- 0.3 s (approximately 15 beats) of the onset of illumination. All hearts developed ventricular premature beats, ventricular tachycardia and complete atrioventricular block after 20.2 +/- 6.6, 68.0 +/- 29.7 and 184.3 +/- 20.9 s, respectively. Photoactivation by rose bengal also resulted in severe ultrastructural damage including intracellular clarifications, swelling of mitochondria with disruption and clumping of cristae and the development of contraction band necrosis. Extensive degranulation of mast cells was also observed. These changes were most evident in myocytes adjacent to large epicardial blood vessels. Cytochemical studies demonstrated that there was a loss of the calcium which is normally localized at the inner sarcolemmal surface, and the appearance of intramitochondrial calcium precipitates. In control hearts (no illumination and/or no rose bengal), arrhythmias did not develop and tissue morphology and calcium distribution remained normal. In additional studies, rose bengal-perfused hearts were illuminated regionally for 10 min over an area (approximately 6 mm2) of the left ventricle. Extensive tissue injury and calcium overload developed in the area of maximum illumination.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Mitochondrial hydrogen peroxide generation by NADH-oxidase activity following regional myocardial ischemia in the dog.

Recently, an exogenous NADH-oxidase has been shown to be a source of oxygen derived toxic species in heart mitochondria. This enzyme uses NADH and oxygen to form superoxide radicals and hydrogen peroxide. Growing evidence suggests that oxygen radicals and hydrogen peroxide may contribute to cardiac damage during ischemia or hypoxia. The activity of the enzyme NADH-oxidase could play an important role in the damage caused by oxygen derived toxic species, especially since cellular defense mechanisms against free radicals are depleted under ischemic conditions. In this study, a cytochemical method was used to visualize hydrogen peroxide, the reaction product of NADH-oxidase activity, in normal and ischemic dog myocardium. The NADH-oxidase reaction product was present in weak amounts in mitochondria from normoxic myocardium. In viable ischemic areas a high degree of activity was observed in the mitochondria. In infarcted tissue mitochondria contained few or no reaction product at all. The results support the hypothesis that hydrogen peroxide and oxygen radicals produced in the mitochondria by a high NADH-oxidase activity may contribute to the mitochondrial damage observed during ischemia when NADH is no longer oxidized by the respiratory chain and cellular defense mechanisms are impaired.

Animals↗

PubMed1988

Purine nucleoside phosphorylase: a histochemical marker for glial cells.

The distribution of purine nucleoside phosphorylase activity has been investigated histochemically in rat and guinea-pig brain. At the light microscopical level, enzyme activity was most pronounced in glial cells in various anatomical regions of the rat brain. In contrast, the guinea-pig brain presented only a weak activity. Endothelial cells of both species were also reactive. These findings were confirmed by electron microscopy. Based upon anatomical position and morphologic characteristics, positive glial cells were identified as astrocytes. Precipitate-rich astrocytic processes could be easily demonstrated in between barely reactive neuronal fibers and around microvessels. A minority of astrocytes was devoid of reaction product. The present method may offer a valuable tool for the histopathological study of several types of disorders in which glial cells play a functional role.

Animals↗

PubMed1988

Ultrastructural localization of calcium in the myocardium of cardiomyopathic syrian hamsters.

Cardiomyopathy of the Syrian hamster is characterized by myocardial calcium overload and focal myocardial necrosis. The cause of the myocardial calcium overload is not yet fully understood. In this study, the ultrastructural localization of calcium was determined in normal hamster hearts and in non-necrotic and necrotic myocardium of cardiomyopathic hamsters (strain BIO 82.62). In many cells from the non-necrotic myocardium of the cardiomyopathic hamsters the calcium deposits, visible as 20 nm particles, were confined to the inner leaflet of the plasma membrane, the T-tubules and the intercalated disks. This corresponds to the calcium distribution found in normal hamsters and other mammalian species. A number of morphologically normal cells, however, displayed an increased amount of calcium precipitate in the mitochondria as well as at the sarcolemma indicating that, in the cardiomyopathic hamster, focal calcium overload is detectable cytochemically in cells which otherwise do not show gross abnormalities. In cells showing morphological signs of myolytic degeneration a marked redistribution of calcium precipitate took place. Sarcolemma became devoid of calcium deposits whereas an enormous amount of clustered precipitate occurred in largely swollen mitochondria. These data are in support of a relationship between impaired ion homeostasis and degeneration events in cardiomyopathy. Furthermore, there appears to be a clear parallelism in calcium redistribution between hypoxic and cardiomyopathic myocardium.

Animals↗

PubMed1985

Sarcolemma-bound calcium. Its importance for cell viability.

We report the presence of a sarcolemma-associated Ca2+-pool in the intact myocardium and isolated ventricular myocytes of the rat. Ca2+-deposits, which are visualized in the electron microscope as 20 nm thick particles, could be demonstrated when fixation of the tissue was performed in the presence of high inorganic phosphate and most probably represent stable Ca2+-acidic phospholipid-phosphate complexes. Various pathophysiological conditions all leading to intracellular Ca2+-overload and compromising myocardial cell viability were imposed and the distribution of Ca2+ was assessed. It was found that in all these conditions cellular Ca2+-overload was preceded or at least accompanied by a loss of sarcolemma-associated Ca2+-deposits. It is concluded that this Ca2+, possibly bound to acidic phospholipids of the sarcolemmal bilayer, plays a role in the maintenance of sarcolemmal integrity. These cytological observations support a concept previously proposed by Langer and his group on the controlling role of membrane Ca2+ in the overall cellular Ca2+-homeostasis.

Animals↗

PubMed1978

Alkaline phosphatase activity in human polymorphonuclear leukocytes.

Alkaline phosphatase has been localized ultracytochemically in PMN of man with normal and elevated levels of this enzyme. Contrary to guinea-pig PMN, no activity appears to be present in the specific granules. Instead, the plasma membrane and the membrane of the endocytic vacuoles show a strong staining. However, the demonstration of this activity depends on the preparatory procedure employed for PMN isolation. The use of dextran and Ficoll-Hypaque in the isolation procedure induces a marked increase in alkaline phosphatase staining of the PMN plasma membrane. Strongly increased activity at this site has been found in PMN from cancer patients. In most of them, additional staining has been observed in atypical vesicles and sometimes in the Golgi apparatus. These findings are discussed in the light of some previously reported controversial biochemical and cytochemical data on the distribution of alkaline phosphatase in human PMN.

Alkaline Phosphatase↗

PubMed1978

Purine nucleoside phosphorylase in chronic lymphocytic leukemia (CLL).

Purine nucleoside phosphorylase (PNP), the enzyme schematically next to adenosine deaminase in the purine salvage pathway, has been demonstrated cytochemically in peripheral blood lymphocytes of healthy subjects and chronic lymphocytic leukemia (CLL) patients. The enzyme activity is confined to the cytosol. In healthy subjects the majority of lymphocytes are strongly reactive for PNP, whereas the rest are devoid of cytochemically demonstrable activity. The percentage of PNP-positive cells largely corresponds to the number of E rosette-forming cells and is inversely proportional to the number of Ig-bearing cells. In six of seven CLL patients studied only a minor percentage of the lymphocytes showed strong PNP activity, whereas the large majority (88%--98%) possessed trace activity. Such patients have a high number of Ig-bearing cells and a low number of E rosette-forming cells. A different pattern of markers was found in the lymphocytes of the seventh CLL patient: 66% were strongly reactive for PNP, an important number formed E rosettes, and a minor percentage were Ig bearing. These data indicate that PNP can be useful as a "nonmembrane" marker in the differentiation of the B and T cell origin in CLL and deserves to be studied in other lymphoproliferative disorders.

Histocytochemistry↗

PubMed1977

Purine nucleoside phosphorylase, a possible histochemical marker for T-cells in man.

A procedure is described for the histochemical detection of purine nucleoside phosphorylase (PNP) activity in circulating lymphocytes of man. The number of PNP-positive cells, as evaluated on smears of Ficoll--Hypaque purified cells, correlated well with the number of E-rosette-forming cells of the same blood samples of healthy and diseased people with normal or abnormal numbers of E-rosettes. In healthy people, the number of PNP-positive cells was within the range of 70-80% of the total lymphocyte population, whilst the corresponding E-rosette-forming cells were scored between 60-75%. Patients with unusually low or high E-rosettes had equally low or high numbers of PNP-reactive cells. More substantial evidence for the presence of PNP activity in T-cells and not in B cells was gathered from experiments in which PNP activity and surface membrane immunoglobulins (SMIg) were simultaneously demonstrated on the same preparation. These results showed, on the one hand, that the bulk of lymphocytes that are reactive for PNP do not reveal SMIg and, on the other hand, that most Ig-bearing cells were unreactive for PNP.

Histocytochemistry↗

PubMed1977

Cytochemical localization of NADH oxidase in Candida albicans.

The application of a recently published technique to localize reduced nicotinamide adenine dinucleotide oxidase activity is described in glutaraldehyde-fixed Candida albicans. The reaction product appears as a finely granular precipitate on the mitochondrial cristae and on the central vacuolar membrane, and, if present, on the vacuolar contents. Fixation should be kept to a minimum and prolonged incubation times up to 2 hr are necessary to show these reactive sites. The reaction appears to be strongly substrate-dependent and not affected by cyanide. Exposure of C. albicans cells to the antimycotic miconazole resulted in a strong increase in reduced nicotinamide, adenine dinucleotide and oxidase activity. The hypothesis is put forward that this enzyme, together with peroxidative and catalatic enzymes, may be implicated in the mechanism by which miconazole exerts its lethal effect on C. albicans.

Candida albicans↗

PubMed1976

Further characterization of phosphatase activities using non-specific substrates.

The demonstration of non-lysosomal acid phosphatase has been the subject of a number of recent investigations. In the present study we compared the enzyme activities in rat liver and kidney that are revealed after incubation in the presence of either beta-glycerophosphate, p-nitrophenylphosphate or phenylphosphate at varying pH. As seen by others, the activity towards p-nitrophenylphosphate at pH 5-6 was confined to lysosomes, Golgi apparatus, endoplasmic reticulum (ER), nuclear envelope and plasmalemma. The reactivity of the plasmalemma and the ER was increased at pH 7. The TER of Küpffer cells in the liver stained intensely in contrast to the ER of the parenchymal cells, which stained only weakly. In the presence of NaF, all sites except the plasmalemma became negative. Addition of a levamisole-analogue, L-p-bromotetramisole, which is a specific inhibitor of alkaline phosphatase, resulted in the disappearance of the plasmalemmal activity whereas the activity at the other sites appeared unaltered. The rather unusual locations of activities with so-called non-specific substrates were further compared with those obtained with specific substrates such as glucose-6-phosphate and thiamine pyrophospate. The possible implication of these data in relation to the specificity of marker-enzymes for subcellular organelles is discussed.

Animals↗

PubMed1975

The inhibition of alkaline phosphatase by L-p-bromotetramisole.

A levamisole analogue, the L-p-bromotetramisole is introduced as a potent inhibitor of non-specific alkaline phosphatase. Complete inhibition is achieved cytochemically at a concentration of 0.1 mM in various rat tissues except the intestine, which is not affected. The D-p-bromotetramisole does not influence the alkaline phosphatase activities. Since no effect of the inhibitor is seen on the activities of specific phosphatases, this drug is recommended also as an additive for specific phosphatase media in order to yield the specific activity only.

Adenosine Triphosphatases↗

PubMed1975

The development of alkaline phosphatase in trichinous muscle.

The development of alkaline phosphatase during invasion and encystment of Trichinella spiralis in rat skeletal muscle fibres was studied at the ultrastructural level. On day 14 after infection, the enzymatic activity is found in proliferating parts of the T-tubular system and in parts of the plasmalemma. In cells, in which a strong hyperplasia of this system is noted. AlPase is present in the abundant network of stratified and concentric membranes from which a large number of pinocytic vesicles arise. From day 50 till 1 year after infection the enzyme activity was invariably present in the matrix surrounding the larvae and was confined to the enormous amounts of cytoplasmic membranes. The possible functional significance of this enzyme in the matrix, in view of its peculiar localization in the immediate vicinity of the parasite, is discussed. In the presence of 0.1 mM of the levamisole analogue, compound R 30402, which is a stereospecific inhibitor of AlPase, the activity is completely lost.

Alkaline Phosphatase↗

PubMed1975

Enzyme cytochemistry of Candida albicans.

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.

Acid Phosphatase↗